Alpha 1-3 N-galactosylstransferase with altered donor specificities, compositions and methods of use

ABSTRACT

The invention generally features compositions and methods based on the structure-based design of alpha 1-3 N-Acetylgalactosaminyltransferase (alpha 3 GalNAc-T) enzymes from alpha 1-3galactosyltransferase (a3Gal-T) that can transfer 2′-modified galactose from the corresponding UDP-derivatives due to substitutions that broaden the alpha 3Gal-T donor specificity and make the enzyme a3 GalNAc-T.

RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE

This application is a Continuation of U.S. application Ser. No.12/674,638, filed Feb. 22, 2010 and to be issued as U.S. Pat. No.8,425,901, which is a U.S. National Phase Application pursuant to 35U.S.C. §371, of PCT International Application Ser. No.PCT/US2007/018678, filed Aug. 22, 2007, designating the United Statesand published in English on Feb. 26, 2009 as publication WO 2009/025646A1. The entire disclosure of the aforementioned patent applications areincorporated herein by this reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Research supporting this application was carried out by the UnitedStates of America as represented by the Secretary, Department of Healthand Human Services. This research was supported [in part] by theIntramural Research Program of the NIH, National Cancer Institute,Center for Cancer Research. This research has been funded in part withFederal funds from the National Cancer Institute, NIH, under contractNo. N01-C0-12400. The Government may have certain rights in thisinvention.

FIELD OF THE INVENTION

The invention relates generally to the structure-based design of alpha1-3 N-Acetylgalactosaminyltransferase (alpha 3 GalNAc-T) enzymes fromalpha 1-3 galactosyltransferase (a3Gal-T). The novel alpha 1-3GalNAc-transferases described herein can transfer 2′-modified galactosefrom the corresponding UDP-derivatives due to substitutions that broadenthe alpha 3Gal-T donor specificity and make the enzyme a3 GalNAc-T.

BACKGROUND OF THE INVENTION

The present invention relates to the field of glycobiology, andspecifically to glycosyltransferases, a superfamily of enzymes that areinvolved in synthesizing the carbohydrate moieties of glycoproteins,glycolipids and glycosaminoglycans. The present invention provides thestructure-based design of novel glycosyltransferases and theirbiological applications.

Glycans can be classified as linear or branched sugars. The linearsugars are the glycosaminoglycans comprising polymers of sulfateddisaccharide repeat units that are O-linked to a core protein, forming aproteoglycan aggregate (Raman et al. 2005). The branched glycans arefound as N-linked and O-linked sugars on glycoproteins or on glycolipids(Lowe et al., 2003). These carbohydrate moieties of the linear andbranched glycans are synthesized by a super family of enzymes, theglycosyltransferases (GTs), which transfer a sugar moiety from a sugardonor to an acceptor molecule. Although GTs catalyze chemically similarreactions in which a monosaccharide is transferred from an activatedderivative, such as a UDP-sugar, to an acceptor, very few GTs bearsimilarity in primary structure.

Eukaryotic cells express several classes of oligosaccharides attached toproteins or lipids. Animal glycans can be N-linked via beta-GlcNAc toAsn (N-glycans), O-linked via -GalNAc to Ser/Thr (O-glycans), or canconnect the carboxyl end of a protein to a phosphatidylinositol unit(GPI-anchors) via a common core glycan structure.

The structural information of glycosyltransferases has revealed that thespecificity of the sugar donor in these enzymes is determined by a fewresidues in the sugar-nucleotide binding pocket of the enzyme, which isconserved among the family members from different species. Thisconservation has made it possible to reengineer the existingglycosyltransferases with broader sugar donor specificities. Mutation ofthese residues generates novel glycosyltransferases that can transfer asugar residue with a chemically reactive functional group toN-acetylglucosamine (GlcNAc), galactose (Gal) and xylose residues ofglycoproteins, glycolipids and proteoglycans (glycoconjugates). Thus,there is potential to develop mutant glycosyltransferases to produceglycoconjugates carrying sugar moieties with reactive groups that can beused in the assembly of bio-nanoparticles to develop targeted-drugdelivery systems or contrast agents for medical uses.

Accordingly, methods to synthesize N-acetylglucosamine linkages havemany applications in research and medicine, including in the developmentof pharmaceutical agents and improved vaccines that can be used to treatdisease.

SUMMARY OF THE INVENTION

As described below, the present invention features the structure-baseddesign of alpha 1-3 N-Acetylgalactosaminyltransferase (alpha 3 GalNAc-T)enzymes from alpha 1-3galactosyltransferase (a3Gal-T). The novel alpha1-3 GalNAc-transferases described herein can transfer 2′-modifiedgalactose from the corresponding UDP-derivatives due to substitutionsthat broaden the alpha 3Gal-T donor specificity and make the enzyme a3GalNAc-T.

In one aspect the invention provides a polypeptide fragment of an alpha1,3 N-acetylgalactosaminyltransferase (alpha 3GalNaC T) that retains theability to transfer a sugar with a chemically reactive functional groupfrom a sugar donor to a sugar acceptor.

In one embodiment, the polypeptide fragment comprises a donorsubstrate-binding site, a hinge region and a DXD motif. In anotherembodiment the polypeptide fragment comprises one or more substitutionsin the donor substrate-binding site.

In a further embodiment, the polypeptide fragment comprises one or moresubstitutions in the hinge region. In still another embodiment,polypeptide fragment comprises one or more substitutions near the DXDmotif.

In a related embodiment, the one or more substitutions in the substratebinding site comprise an amino acid substitution at position 280, 281,or 282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or moresubstitutions in the substrate hinge region comprise an amino acidsubstitution at position 191 corresponding to bovine alpha 1,3galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In oneembodiment, the one or more substitutions close to the DXD motifcomprise an amino acid substitution at position 228 corresponding tobovine alpha 1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21).In another embodiment, wherein a serine (S) is substituted for ahistidine (H) at amino acid position 280, a glycine (G) is substitutedfor an alanine (A) at amino acid position 281, or a glycine (G) issubstituted for an alanine at amino acid position 282 of (SEQ ID NO 21).In another embodiment, a serine (S) or an alanine (A) is substituted fora proline (P) at amino acid position 191 corresponding to (SEQ ID NO:21). In still another embodiment, a glutamine (Q) is replaced with a amethionine (M) at amino acid position 228 of (SEQ ID NO:21).

In another aspect, the invention features a polypeptide fragment of analpha 1,3 N-acetylgalactosaminyltransferase (alpha3GalNAc-T) thattransfers a sugar with a chemically reactive functional group from asugar donor to a sugar acceptor, wherein the polypeptide fragmentcomprises and one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, or SEQ ID NO: 20

In one embodiment of the above-mentioned aspects, the sugar acceptor isselected from the group consisting of galactose beta 1,4 GlcNac andgalactose beta 1,4 glucose.

In another embodiment of the above-mentioned aspects, the sugar with achemically reactive functional group is selected from the groupconsisting of UDP-GalNAc, UDP-galactose, and UDP-galactose analogues.

In one embodiment, the UDP-galactose analogue comprises an azido group,a keto group or a thiol group.

In another embodiment, the azido group, the keto group or the thiolgroup is substituted at the C2 position of galactose. In a furtherembodiment, the one or more agents are linked to a sugar moiety of thesugar donor. In one embodiment, the one or more agents is selected fromthe group consisting of: single chain antibodies, bacterial toxins,growth factors, therapeutic agents, targeting agents, contrast agents,chemical labels, radiolabels, and fluorescent labels.

In another aspect, the invention features a polypeptide fragment of analpha 1,3 N-Acetylgalactosaminyltransferase (alpha3GalNac-T) thatretains that ability to transfer a sugar with a chemically reactivefunctional group from a sugar donor to a sugar acceptor and catalyzesthe formation of an oligosaccharide. In one embodiment, theoligosaccharide is a disaccharide or a trisaccharide. In anotherembodiment, the trisaccharide is selected from the group consisting of:GalNAc alpha1-3Galbeta 1-4Gal, GalNAc alpha1-3-Galbeta 1-4GlcNAc,2′-modified-Gal alpha 1-3 Gal beta 1-4Gal or 2′-modified-Galalpha1-3-Gal beta 1-4GlcNAc. In one embodiment, the polypeptide fragmentcomprises any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, or SEQ ID NO: 20.

In another aspect, the invention features an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 60%homologous to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO; 11, SEQ ID NO: 13, SEQID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19 or a complement thereof.

In still another aspect, the invention features an isolated nucleic acidmolecule which encodes a polypeptide comprising an amino acid sequenceat least about 50% homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO;12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

In one embodiment, the expression cassette or vector comprises thenucleic acid of the aspects as described herein.

In another aspect, the invention features an expression cassette orvector comprising a nucleic acid segment encoding a polypeptide fragmentfrom an alpha 1,3 N acetylgalactosaminyltransferase (alpha 3Gal NAcTthat transfers a sugar with a chemically reactive functional group froma sugar donor to a sugar acceptor.

In one embodiment, the cell comprises the expression cassette or vectoras described in the aspects herein.

In another aspect, the invention features a method to of making anoligosaccharide comprising incubating a reaction mixture comprising apolypeptide fragment of an alpha 1,3 N-acetylgalactosaminyltransferase(alpha 3GalNaC T) that retains the ability to transfer a sugar with achemically reactive functional group with a sugar donor and a sugaracceptor.

In one embodiment, the polypeptide fragment comprises a donor substratebinding site, a hinge region and a DXD motif. In another embodiment, thepolypeptide fragment comprises one or more substitutions in the donorsubstrate binding site. In another embodiment, the polypeptide fragmentcomprises one or more substitutions in the hinge region. In anotherembodiment, the polypeptide fragment comprises one or more substitutionsnear the DXD motif.

In a related embodiment, the one or more substitutions in the substratebinding site comprise an amino acid substitution at position 280, 281,or 282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or moresubstitutions in the substrate hinge region comprise an amino acidsubstitution at position 191 corresponding to bovine alpha 1,3galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In oneembodiment, the one or more substitutions close to the DXD motifcomprise an amino acid substitution at position 228 corresponding tobovine alpha 1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21).In another embodiment, wherein a serine (S) is substituted for ahistidine (H) at amino acid position 280, a glycine (G) is substitutedfor an alanine (A) at amino acid position 281, or a glycine (G) issubstituted for an alanine at amino acid position 282 of (SEQ ID NO 21).In another embodiment, a serine (S) or an alanine (A) is substituted fora proline (P) at amino acid position 191 corresponding to (SEQ ID NO:21). In still another embodiment, a glutamine (Q) is replaced with amethionine (M) at amino acid position 228 of (SEQ ID NO:21).

In another aspect, the invention features a method of making anoligosaccharide comprising incubating a reaction mixture comprising apolypeptide fragment of an alpha 1,3 N-acetylgalactosaminyltransferase(alpha 3GalNaC T) that retains the ability to transfer a sugar with achemically reactive functional group with a sugar donor and a sugaracceptor, wherein the polypeptide fragment comprises any one of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

In one embodiment, the sugar acceptor is selected from the groupconsisting of galactose beta 1,4 GlcNac and galactose beta 1,4 glucose.

In another embodiment, the sugar with a chemically reactive functionalgroup is selected from the group consisting of UDP-GalNAc,UDP-galactose, and UDP-galactose analogues.

In a further embodiment, the UDP-galactose analogue comprises an azidogroup, a keto group or a thiol group. In another embodiment, the azidogroup, the keto group or the thiol group is substituted at the C2position of galactose. In still a further embodiment, one or more agentsare linked to a sugar moiety of the sugar donor.

In one embodiment, the one or more agents is selected from the groupconsisting of: single chain antibodies, bacterial toxins, growthfactors, therapeutic agents, targeting agents, contrast agents, chemicallabels, radiolabels, and fluorescent labels.

In another embodiment, the oligosaccharide is a disaccharide or atrisaccharide. In a related embodiment, the trisaccharide is selectedfrom the group consisting of: GalNAc alpha1-3Galbeta 1-4Gal, GalNAcalpha1-3-Galbeta 1-4GlcNAc, 2′-modified-Gal alpha 1-3 Gal beta 1-4Gal or2′-modified-Galalpha 1-3-Gal beta 1-4GlcNAc.

In one embodiment, the polypeptide fragment comprises any one of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

In another aspect, the invention features an oligosaccharide synthesizedby the method comprising incubating a reaction mixture comprising apolypeptide fragment of an alpha 1,3 N-acetylgalactosylaminotransferase(alpha 3GalNAc T) that retains the ability to transfer a sugar with achemically reactive functional group with a sugar donor and a sugaracceptor.

In one embodiment, the polypeptide fragment comprises a donor substratebinding site, a hinge region and a DXD motif. In another embodiment, thepolypeptide fragment comprises one or more substitutions in the donorsubstrate binding site. In one embodiment, the polypeptide fragmentcomprises one or more substitutions in the hinge region. In oneembodiment, the polypeptide fragment comprises one or more substitutionsnear the DXD motif.

In a related embodiment, the one or more substitutions in the substratebinding site comprise an amino acid substitution at position 280, 281,or 282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or moresubstitutions in the substrate hinge region comprise an amino acidsubstitution at position 191 corresponding to bovine alpha 1,3galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In oneembodiment, the one or more substitutions close to the DXD motifcomprise an amino acid substitution at position 228 corresponding tobovine alpha 1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21).In another embodiment, wherein a serine (S) is substituted for ahistidine (H) at amino acid position 280, a glycine (G) is substitutedfor an alanine (A) at amino acid position 281, or a glycine (G) issubstituted for an alanine at amino acid position 282 of (SEQ ID NO 21).In another embodiment, a serine (S) or an alanine (A) is substituted fora proline (P) at amino acid position 191 corresponding to (SEQ ID NO:21). In still another embodiment, a glutamine (Q) is replaced with amethionine (M) at amino acid position 228 of (SEQ ID NO:21).

In another aspect, the invention features an oligosaccharide synthesizedby the method comprising incubating a reaction mixture comprising apolypeptide fragment of an alpha 1,3 N-Acetylgalactosylaminotransferase(alpha 3GalNaC T) that retains the ability to transfer a sugar with achemically reactive functional group with a sugar donor and a sugaracceptor, wherein the polypeptide fragment comprises any one of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

In one embodiment, the sugar acceptor is selected from the groupconsisting of galactose beta 1,4 GlcNAc and galactose beta 1,4 glucose.In another embodiment, the sugar with a chemically reactive functionalgroup is selected from the group consisting of UDP-GalNAc,UDP-galactose, and UDP-galactose analogues.

In another embodiment, the UDP-galactose analogue comprises an azidogroup, a keto group or a thiol group. In a related embodiment, the azidogroup, the keto group or the thiol group is substituted at the C2position of galactose.

In a related embodiment, one or more agents are linked to a sugar moietyof the sugar donor. In another embodiment, the one or more agents isselected from the group consisting of: single chain antibodies,bacterial toxins, growth factors, therapeutic agents, targeting agents,contrast agents, chemical labels, radiolabels, and fluorescent labels.

In one embodiment, the oligosaccharide is a disaccharide or atrisaccharide. In another embodiment, the trisaccharide is selected fromthe group consisting of: GalNAc alpha1-3Galbeta 1-4Gal, GalNAcalpha1-3-Galbeta 1-4GlcNAc, 2′-modified-Gal alpha 1-3 Gal beta 1-4Gal or2′-modified-Galalpha 1-3-Gal beta 1-4GlcNAc.

In another embodiment of the above-mentioned aspects, the polypeptidefragment comprises any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO; 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, or SEQ ID NO: 20.

In another aspect, the invention features a composition comprising apolypeptide fragment of an alpha 1,3 N-Acetylgalactosylaminotransferase(alpha 3GalNaC T) that retains the ability to transfer a sugar with achemically reactive functional group from a sugar donor to a sugaracceptor.

In one embodiment, the polypeptide fragment comprises a donor substratebinding site, a hinge region and a DXD motif. In another embodiment, thepolypeptide fragment comprises one or more substitutions in the donorsubstrate binding site. In another embodiment, the polypeptide fragmentcomprises one or more substitutions in the hinge region. In anotherembodiment, the polypeptide fragment comprises one or more substitutionsnear the DXD motif.

In a related embodiment, the one or more substitutions in the substratebinding site comprise an amino acid substitution at position 280, 281,or 282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or moresubstitutions in the substrate hinge region comprise an amino acidsubstitution at position 191 corresponding to bovine alpha 1,3galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In oneembodiment, the one or more substitutions close to the DXD motifcomprise an amino acid substitution at position 228 corresponding tobovine alpha 1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21).In another embodiment, wherein a serine (S) is substituted for ahistidine (H) at amino acid position 280, a glycine (G) is substitutedfor an alanine (A) at amino acid position 281, or a glycine (G) issubstituted for an alanine at amino acid position 282 of (SEQ ID NO 21).In another embodiment, a serine (S) or an alanine (A) is substituted fora proline (P) at amino acid position 191 corresponding to (SEQ ID NO:21). In still another embodiment, a glutamine (Q) is replaced with a amethionine (M) at amino acid position 228 of (SEQ ID NO:21).

In another aspect, the invention features a composition comprising apolypeptide fragment of an alpha 1,3 N-Acetylgalactosaminyltransferase(alpha3GalNac-T) that transfers a sugar with a chemically reactivefunctional group from a sugar donor to a sugar acceptor, wherein thepolypeptide fragment comprises any one of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO; 12, SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

In one embodiment, the sugar acceptor is selected from the groupconsisting of galactose beta 1,4 GlcNAc and galactose beta 1,4 glucose.In another embodiment, the sugar with a chemically reactive functionalgroup is selected from the group consisting of UDP-GalNAc,UDP-galactose, and UDP-galactose analogues. In another embodiment, theUDP-galactose analogue comprises an azido group, a keto group or a thiolgroup. In another embodiment, the keto group or the thiol group issubstituted at the C2 position of galactose. In another embodiment, oneor more agents are linked to a sugar moiety of the sugar donor. In arelated embodiment, the one or more agents is selected from the groupconsisting of: single chain antibodies, bacterial toxins, growthfactors, therapeutic agents, targeting agents, contrast agents, chemicallabels, radiolabels, and fluorescent labels.

In another aspect, the invention features a composition comprising apolypeptide fragment of an alpha 1,3 N-Acetylgalactosaminyltransferase(alpha3GalNac-T) that retains that ability to transfer a sugar with achemically reactive functional group from a sugar donor to a sugaracceptor and catalyzes the formation of an oligosaccharide.

In one embodiment, the oligosaccharide is a disaccharide or atrisaccharide. In another embodiment, the trisaccharide is selected fromthe group consisting of: GalNAc alpha1-3Galbeta 1-4Gal, GalNAcalpha1-3-Galbeta 1-4GlcNAc, 2′-modified-Gal alpha 1-3 Gal beta 1-4Gal or2′-modified-Galalpha 1-3-Gal beta 1-4GlcNAc.

In another embodiment, the polypeptide fragment comprises any one of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO:20.

In another aspect, the invention features an immunological compositioncomprising a polypeptide fragment of an alpha 1,3N-Acetylgalactosylaminotransferase (alpha 3GalNaC T) that retains theability to transfer a sugar with a chemically reactive functional groupfrom a sugar donor to a sugar acceptor.

In one embodiment, the polypeptide fragment comprises a donorsubstrate-binding site, a hinge region and a DXD motif. In anotherembodiment, the polypeptide fragment comprises one or more substitutionsin the donor substrate-binding site. In another embodiment, thepolypeptide fragment comprises one or more substitutions in the hingeregion. In another embodiment, the polypeptide fragment comprises one ormore substitutions near the DXD motif.

In a related embodiment, the one or more substitutions in the substratebinding site comprise an amino acid substitution at position 280, 281,or 282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or moresubstitutions in the substrate hinge region comprise an amino acidsubstitution at position 191 corresponding to bovine alpha 1,3galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In oneembodiment, the one or more substitutions close to the DXD motifcomprise an amino acid substitution at position 228 corresponding tobovine alpha 1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21).In another embodiment, wherein a serine (S) is substituted for ahistidine (H) at amino acid position 280, a glycine (G) is substitutedfor an alanine (A) at amino acid position 281, or a glycine (G) issubstituted for an alanine at amino acid position 282 of (SEQ ID NO 21).In another embodiment, a serine (S) or an alanine (A) is substituted fora proline (P) at amino acid position 191 corresponding to (SEQ ID NO:21). In still another embodiment, a glutamine (Q) is replaced with amethionine (M) at amino acid position 228 of (SEQ ID NO:21).

In another aspect, the invention features an immunological compositioncomprising a polypeptide fragment of an alpha 1,3N-Acetylgalactosaminyltransferase (alpha3GalNac-T) that transfers asugar with a chemically reactive functional group from a sugar donor toa sugar acceptor, wherein the polypeptide fragment comprises and one ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ IDNO: 20, and wherein one or more antibodies are conjugated to thechemically reactive functional group.

In one embodiment, the sugar acceptor is selected from the groupconsisting of galactose beta 1,4 GlcNac and galactose beta 1,4 glucose.In another embodiment, the sugar with a chemically reactive functionalgroup is selected from the group consisting of UDP-GalNAc,UDP-galactose, and UDP-galactose analogues. In another embodiment, theUDP-galactose analogue comprises an azido group, a keto group or a thiolgroup. In another further embodiment, the azido group, the keto group orthe thiol group is substituted at the C2 position of galactose.

In another embodiment, one or more agents are linked to a sugar moietyof the sugar donor. In another embodiment, the agent is selected fromsingle chain antibodies, monoclonal antibodies, polyclonal antibodies,and chimeric antibodies.

In another aspect, the invention features an immunological compositioncomprising a polypeptide fragment of an alpha 1,3N-Acetylgalactosaminyltransferase (alpha3GalNac-T) that retains thatability to transfer a sugar with a chemically reactive functional groupfrom a sugar donor to a sugar acceptor and catalyzes the formation of anoligosaccharide, and wherein one or more antibodies are conjugated tothe chemically reactive functional group.

In one embodiment, the oligosaccharide is a disaccharide or atrisaccharide. In a related embodiment, the trisaccharide is selectedfrom the group consisting of: GalNAc alpha1-3Galbeta 1-4Gal, GalNAcalpha1-3-Galbeta 1-4GlcNAc, 2′-modified-Gal alpha 1-3 Gal beta 1-4Gal or2′-modified-Galalpha 1-3-Gal beta 1-4GlcNAc. In another embodiment, thepolypeptide fragment comprises any one of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO; 12, SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

In another aspect the invention features a method of coupling an agentto a carrier protein comprising incubating a reaction mixture comprisingA polypeptide fragment of an alpha 1,3N-Acetylgalactosylaminotransferase (alpha 3GalNaC T) that retains theability to transfer a sugar with a chemically reactive functional groupfrom a sugar donor to a sugar acceptor, wherein the sugar donor iscoupled to an agent and the sugar acceptor is a carrier protein.

In another embodiment the polypeptide fragment comprises a donorsubstrate binding site, a hinge region and a DXD motif. In anotherembodiment, the polypeptide fragment comprises one or more substitutionsin the donor substrate-binding site. In another embodiment, thepolypeptide fragment comprises one or more substitutions in the hingeregion. In another embodiment, the polypeptide fragment comprises one ormore substitutions near the DXD motif.

In a related embodiment, the one or more substitutions in the substratebinding site comprise an amino acid substitution at position 280, 281,or 282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or moresubstitutions in the substrate hinge region comprise an amino acidsubstitution at position 191 corresponding to bovine alpha 1,3galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In oneembodiment, the one or more substitutions close to the DXD motifcomprise an amino acid substitution at position 228 corresponding tobovine alpha 1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21).In another embodiment, wherein a serine (S) is substituted for ahistidine (H) at amino acid position 280, a glycine (G) is substitutedfor an alanine (A) at amino acid position 281, or a glycine (G) issubstituted for an alanine at amino acid position 282 of (SEQ ID NO 21).In another embodiment, a serine (S) or an alanine (A) is substituted fora proline (P) at amino acid position 191 corresponding to (SEQ ID NO:21). In still another embodiment, a glutamine (Q) is replaced with a amethionine (M) at amino acid position 228 of (SEQ ID NO:21).

In another embodiment, the sugar acceptor is selected from the groupconsisting of galactose beta 1,4 GlcNac and galactose beta 1,4 glucose.

In another embodiment, the sugar with a chemically reactive functionalgroup is selected from the group consisting of UDP-GalNAc,UDP-galactose, and UDP-galactose analogues. In a related embodiment, theUDP-galactose analogue comprises an azido group, a keto group or a thiolgroup. In another related embodiment, the azido group, the keto group orthe thiol group is substituted at the C2 position of galactose.

In another embodiment, one or more agents are linked to a sugar moietyof the sugar donor.

In another embodiment, the one or more agents is selected from the groupconsisting of: single chain antibodies, bacterial toxins, growthfactors, therapeutic agents, targeting agents, contrast agents,paramagnetic contrast agents, chemical labels, radiolabels, andfluorescent labels.

In one embodiment, the oligosaccharide is a disaccharide or atrisaccharide. In another embodiment, the trisaccharide is selected fromthe group consisting of: GalNAc alpha1-3Galbeta 1-4Gal, GalNAcalpha1-3-Galbeta 1-4GlcNAc, 2′-modified-Gal alpha 1-3 Gal beta 1-4Gal or2′-modified-Galalpha 1-3-Gal beta 1-4GlcNAc.

In another embodiment, the polypeptide fragment comprises any one of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO:20.

In one embodiment, the carrier protein is ovalbumin.

In another embodiment, the carrier protein is an IgG.

In a related embodiment, the method of any of the above-mentionedaspects is used in imaging.

In another related embodiment, the agent is a paramagnetic imaging agentused in magnetic resonance imaging.

In another aspect, the invention features a method for the diagnosis ortreatment of a subject suffering from a disease or disorder comprisingadministering to the subject an effective amount of an isolatedglycoprotein synthesized by the method according to any one theabove-mentioned aspects, wherein one or more agents are linked to thesugar donor, and thereby diagnosing or treating a subject suffering froma disease or disorder.

In one embodiment, the disease or disorder is selected from the groupconsisting of: proliferative diseases, cardiovascular diseases,inflammatory diseases, cancer, diseases of ageing, and metabolicdiseases or disorders.

In another embodiment, the agent is selected from the group consistingof: single chain antibodies, bacterial toxins, growth factors,therapeutic agents, contrast agents, targeting agents, chemical labels,radiolabels, and fluorescent labels.

In another aspect, the invention features a method for imaging a targetcell or tissue comprising administering to a subject an oligosaccharidesynthesized by the method according to any one of the above-mentionedaspects, and wherein one or more imaging agents are linked to the sugardonor, thereby imaging a target cell or tissue.

In still another aspect, the invention features a method forsynthesizing a detectable Gal beta 1-4GlcNAc epitope comprisingsynthesizing an oligosaccharide according to the method of any one ofthe above-mentioned aspects, wherein the sugar donor comprises a 2′modified Gal residue and wherein or more detection agents are linked tothe 2′ modified Gal residue and thereby synthesizing a detectable Galbeta 1-4GlcNAc epitope.

In one embodiment, the detectable Gal beta 1-4GlcNAc epitope isadministered to a subject.

In another embodiment, the detectable Gal beta 1-4GlcNAc epitope isadministered to a subject to diagnose a disease or disorder.

In another embodiment, the disease or disorder selected from the groupconsisting of: proliferative diseases, cardiovascular diseases,inflammatory diseases, cancer, diseases of ageing, and metabolicdiseases or disorders.

In another aspect the invention features a method for inducing an immuneresponse in a subject comprising administering to the subject animmunological composition according to any one of the above-mentionedaspects.

In another aspect, the invention provides a kit comprising packagingmaterial, and an polypeptide fragment from an alpha 1,3N-Acetylgalactosaminyltransferase (alpha3GalNac-T) according to any oneof the above-mentioned aspects.

In one embodiment, the kit comprises a sugar donor.

In another embodiment, the donor is selected from the group consistingof UDP-galactose, UDP-GalNAc or UDP-GalNAc analogue. In a relatedembodiment, an agent is linked to the sugar donor. In another relatedembodiment, the agent is selected from the group consisting of: singlechain antibodies, bacterial toxins, growth factors, therapeutic agents,contrast agents, targeting agents, chemical labels, a radiolabels, andfluorescent labels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the structure—based design ofa1-3-N-acetylgalactosaminyltransferase from a1-3-galactosyltransferase.

FIG. 2 is a diagram illustrating the structure-based design of a1-3N-acetylgalactosaminyltransferase (a3GalNAc-T) froma1-3galactosyltransferase (a3Gal-T). The boxed region shows amagnification of the sugar donor binding site and the hinge region wherethe substitutions occur.

FIG. 3 shows transfer of UDP— modified sugars by the alpha 1,3Gal-T-191A . . . 280SGG282.

FIG. 4 shows transfer of UDP— modified sugars by the alpha 1,3Gal-T-191A . . . 280SGG282.

FIG. 5 is a table showing the effect of substitutions in the donorsubstrate binding site, hinge region and near DXD motif on Gal activity,GalNAc activity and GalKeto activity.

DETAILED DESCRIPTION OF THE INVENTION

The invention generally features compositions and methods based on thestructure-based design of alpha 1-3 N-acetylgalactosaminyltransferase(alpha 3 GalNAc-T) enzymes from alpha 1-3galactosyltransferase (a3Gal-T)that can transfer 2′-modified galactose from the correspondingUDP-derivatives due to substitutions that broaden the alpha 3Gal-T donorspecificity and make the enzyme a3 GalNAc-T.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof. The term “a nucleic acid molecule” includesa plurality of nucleic acid molecules.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude other elements. “Consisting essentially of”, when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants from the isolation and purification methodand pharmaceutically acceptable carriers, such as phosphate bufferedsaline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

The term “acceptor” is meant to refer to a molecule or structure ontowhich a donor is actively linked through action of a catalytic domain ofa galactosyltransferase, or mutant thereof. Examples of acceptorsinclude, but are not limited to, carbohydrates, glycoproteins,glycolipids. The acceptor polypeptide can comprise, in preferredembodiments Galactose residues, free or attached to a peptide orglycopeptide.

The term “agent” or “bioactive agent” is meant to refer to any chemicalor biological material or compound that is suitable for delivery thatinduces a desired effect in or on an organism, such as a biological orpharmacological effect, which may include, but is not limited to aprophylactic effect, alleviating a condition caused by a disease or adisorder, reducing or eliminating a disease or disorder. An agent or abioactive agent refers to substances that are capable of exerting abiological effect in vitro and/or in vivo. Examples include diagnosticagents, pharmaceuticals, drugs, synthetic organic molecules, proteins,peptides, vitamins, steroids, genetic material including nucleotides,nucleosides, polynucleotides, RNAs, siRNAs, shRNAs, anti-sense DNA orRNA.

The term “antibody” as used herein refers to both polyclonal andmonoclonal antibody. The term can also refer to single chain antibodies.The term encompasses not only intact immunoglobulin molecules, butfragments and genetically engineered derivatives of immunoglobulinmolecules as may be prepared by techniques known in the art, and whichretains the binding specificity of the antigen binding site.

The term “polypeptide fragment” refers to an amino acid segment whichfolds into a domain that is able to catalyze the linkage of a donor toan acceptor. A polypeptide fragment may be from any mammalian alpha1-3N-acetylgalactosaminyltransferase (a3 GalNAc-T). In certain embodiments,the polypeptide fragment is from bovine a3 GalNAc-T, in other certainembodiments, the polypeptide fragment is from human a3 GalNAc-T. Inpreferred embodiments, the a3 GalNAc-T polypeptide fragment is selectedfrom the nucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO:3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQID NO: 17, and SEQ ID NO: 19. In other embodiments, the a3 GalNAc-Tpolypeptide fragment is selected from the protein corresponding to theamino acid sequence comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO: 20. The invention relates generally tothe structure-based design of alpha 1-3N-acetylgalactosaminyltransferase (a 3 GalNAc-T) enzymes from alpha1-3galactosyltransferase (a3Gal-T) . . . . ” The polypeptide fragmentused for the construction of a3 GalNAc-T enzymes is, in certainexamples, from bovine alpha-1,3 galactosyltransferase corresponding tothe amino acid sequence NO: 21 as shown herein.

The term “donor” refers to a molecule that is actively linked to anacceptor molecule through the action of a catalytic domain of agalactosyltransferase, or mutant thereof. A donor molecule can include asugar, or a sugar derivative. Examples of donors include, but are notlimited to, UDP-GalNAc, UDP-galactose or UDP-galNAc analogues,UDP-galactose analogues. Donors include sugar derivatives that includeagents, biological agents, or active groups. Accordingly,oligosaccharides may be prepared according to the methods of theinvention that include a sugar derivative having any desiredcharacteristic.

The term “DXD motif” is meant to refer to a glycosyltransferasesugar-binding region containing DXD motif. In preferred embodiments, theDXD motif is a short conserved motif found in many families ofglycosyltransferases, which add a range of different sugars to othersugars, phosphates and proteins. In other certain embodiments,DXD-containing glycosyltransferases all use nucleoside diphosphatesugars as donors and require divalent cations, usually manganese.Preferred DXD motifs are represented by the NCBI conserved domaindatabase designation pfam04488.8.

The term “effective amount” is meant to refer to a sufficient amountthat is capable of providing the desired local or systemic effect.

The term “expression cassette” as used herein refers to a DNA sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest that is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The expression cassette may beone that is naturally occurring but has been obtained in a recombinantform useful for heterologous expression. The expression of thenucleotide sequence in the expression cassette may be under the controlof a constitutive promoter or of an inducible promoter that initiatestranscription only when the host cell is exposed to some particularexternal stimulus. In the case of a multicellular organism, the promotercan also be specific to a particular tissue or organ or stage ofdevelopment.

The term “alpha1-3 N-acetylgalactosaminyltransferase (a3 GalNAc-T)” asused herein refers to enzymes substantially homologous to, and havingsubstantially the same biological activity as, the enzyme coded for bythe nucleotide sequence depicted in any one of SEQ ID NOs: 1, 3, 5, 7,9, 11, 15, 17, 19 and the amino acid sequence depicted in SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20. This definition is intended toencompass natural allelic variations in the a3 GalNAc-T sequence, andall references to a3 GalNAc-T, and nucleotide and amino acid sequencesthereof are intended to encompass such allelic variations, bothnaturally occurring and man-made. The production of proteins such as theenzyme a3 GalNAc-T from cloned genes by genetic engineering is wellknown.

The a3 GalNAc-T enzyme may be synthesized in host cells transformed withvectors containing DNA encoding the a3 GalNAc-T enzyme. A vector is areplicable DNA construct. Vectors are used herein either to amplify DNAencoding the a3 GalNAc-T enzyme and/or to express DNA which encodes thea3 GalNAc-T enzyme. An expression vector is a replicable DNA constructin which a DNA sequence encoding the a3 GalNAc-T enzyme is operablylinked to suitable control sequences capable of effecting the expressionof the a3 GalNAc-T enzyme in a suitable host. The need for such controlsequences will vary depending upon the host selected and thetransformation method chosen. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. Amplification vectors do not require expression controldomains. All that is needed is the ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants.

The term “homologous” is intended to include a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent amino acid residues or nucleotides, e.g., anamino acid residue which has a similar side chain, to a second aminoacid or nucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains and/or a commonfunctional activity.

The terms “oligosaccharide” and “polysaccharide” are usedinterchangeably herein. These terms refer to saccharide chains havingtwo or more linked sugars. Oligosaccharides and polysaccharides may behomopolymers and heteropolymers having a random sugar sequence or apreselected sugar sequence. Additionally, oligosaccharides andpolysaccharides may contain sugars that are normally found in nature,derivatives of sugars, and mixed polymers thereof. “saccharide” refersto any of a series of compounds of carbon, hydrogen, and oxygen in whichthe atoms of the latter two elements are in the ratio of 2:1, especiallythose containing the groupC6H1o05, including fructose, glucose, sucrose,lactose, maltose, galactose and arabinose.

The term “immunogenic” compound or composition as used herein refers toa compound or composition that is capable of stimulating production of aspecific immunological response when administered to a suitable host,usually a mammal.

The term “nucleic acid” is intended to include nucleic acid molecules,e.g., polynucleotides which include an open reading frame encoding apolypeptide, and can further include non-coding regulatory sequences,and introns. In addition, the terms are intended to include one or moregenes that map to a functional locus. In addition, the terms areintended to include a specific gene for a selected purpose. The gene canbe endogenous to the host cell or can be recombinantly introduced intothe host cell, e.g., as a plasmid maintained episomally or a plasmid (orfragment thereof) that is stably integrated into the genome. In oneembodiment, the gene of polynucleotide segment is involved sugartransfer. A mutant nucleic acid molecule or is intended to include anucleic acid molecule or gene having a nucleotide sequence whichincludes at least one alteration (e.g., substitution, insertion,deletion) such that the polypeptide or polypeptide that can be encodedby said mutant exhibits an activity that differs from the polypeptide orpolypeptide encoded by the wild-type nucleic acid molecule or gene.

The terms “polypeptides” or “isolated polypeptide” and “proteins” areused interchangeably herein. Polypeptides and proteins can be expressedin vivo through use of prokaryotic or eukaryotic expression systems.Many such expressions systems are known in the art and are commerciallyavailable. (Clontech, Palo Alto, Calif.; Stratagene, La Jolla, Calif.).Examples of such systems include, but are not limited to, theT7-expression system in prokaryotes and the bacculovirus expressionsystem in eukaryotes. Polypeptides can also be synthesized in vitro,e.g., by the solid phase peptide synthetic method or by in vitrotranscription/translation systems. Such methods are described, forexample, in U.S. Pat. Nos. 5,595,887; 5,116,750; 5,168,049 and5,053,133; Olson et al., Peptides, 9, 301, 307 (1988). The solid phasepeptide synthetic method is an established and widely used method, whichis described in the following references: Stewart et al., Solid PhasePeptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield,J. Am. Chem. Soc., 85 2149 (1963); Meienhofer in “Hormonal Proteins andPeptides,” ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp. 48-267;Bavaay and Merrifield, “The Peptides,” eds. E. Gross and F. Meienhofer,Vol. 2 (Academic Press, 1980) pp. 3-285; and Clark-Lewis et al., Meth.Enzymol., 287, 233 (1997). These polypeptides can be further purified byfractionation on immunoaffinity or ion-exchange columns; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on ananion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;or ligand affinity chromatography. The term an “isolated polypeptide”(e.g., an isolated or purified biosynthetic enzyme) is substantiallyfree of cellular material or other contaminating polypeptides from themicroorganism from which the polypeptide is derived, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized

The polypeptides of the invention include polypeptides having amino acidexchanges, i.e., variant polypeptides, so long as the polypeptidevariant is biologically active. The variant polypeptides include theexchange of at least one amino acid residue in the polypeptide foranother amino acid residue, including exchanges that utilize the Drather than L form, as well as other well known amino acid analogs,e.g., N-alkyl amino acids, lactic acid, and the like. These analogsinclude phosphoserine, phosphothreonine, phosphotyrosine,hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, citruline, N-methyl-alanine, para-benzoyl-phenylalanine,phenylglycine, propargylglycine, sarcosine, N-acetylserine,N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and othersimilar amino acids and imino acids and tert-butylglycine.

Conservative amino acid exchanges are preferred and include, forexample; aspartic-glutamic as acidic amino acids;lysine/arginine/histidine as basic amino acids; leucine/isoleucine,methionine/valine, alanine/valine as hydrophobic amino acids;serine/glycine/alanine/threonine as hydrophilic amino acids.Conservative amino acid exchange also includes groupings based on sidechains. Members in each group can be exchanged with another. Forexample, a group of amino acids having aliphatic side chains is glycine,alanine, valine, leucine, and isoleucine. These may be exchanged withone another. A group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine. A group of amino acids havingamide-containing side chains is asparagine and glutamine. A group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan. A group of amino acids having basic side chains is lysine,arginine, and histidine. A group of amino acids having sulfur-containingside chains is cysteine and methionine. For example, replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid may be accomplished to produce avariant polypeptide of the invention.

The term “subject” as used herein refers to any animal, includingmammals, preferably humans, to which the present invention may beapplied.

The term “cancer” or “tumor” refers to an aggregate of abnormal cellsand/or tissue which may be associated with diseased states that arecharacterized by uncontrolled cell proliferation. The disease states mayinvolve a variety of cell types, including, for example, endothelial,epithelial and myocardial cells. Included among the disease states areneoplasms, cancer, leukemia and restenosis injuries.

Alpha 1,3 N-Acetylgalactosaminyltransferase (a3 GalNAc-T)

Specific glycosyltransferases synthesize oligosaccharides by thesequential transfer of the monosaccharide moiety of an activated sugardonor to an acceptor molecule. Members of the glycosyltransferasesuperfamily, which are often named after the sugar moiety that theytransfer, are divided into subfamilies on the basis of linkage that isgenerated between the donor and acceptor. Transfer of the sugar residueoccurs with either the retention (by retaining glycosyltransferases) orthe inversion (by inverting glycosyltransferases) of the configurationat the anomeric C1 atom. Glycosyltransferases show great structuralsimilarity. They are all globular proteins with two types of fold,termed GT-A and GT-B, which each have an N-terminal and a C-terminaldomain. The enzymes of the GT-A fold have two dissimilar domains. TheN-terminal domain, which recognizes the sugar-nucleotide donor,comprises several b-strands that are each flanked by a-helices as in aRossmann-like fold, whereas the C-terminal domain, which contains theacceptor-binding site, consists largely of mixed b-sheets. By contrast,enzymes with the GT-B fold contain two similar Rossmann-like folds, withthe N-terminal domain providing the acceptor-binding site and theC-terminal domain providing the donor-binding site.

In both types of enzyme, the two domains are connected by a linkerregion and the active site is located between the two domains. Ametal-binding site is also located in the cleft in enzymes of both theGT-B and GT-A fold (Qasba et al. 2005).

Alpha (1,3)-galactosyltransferase I (a3 Gal-T)

The alpha (1,3)-galactosyltransferase I (a3 Gal-T) enzyme mediates theformation of gal-alpha-gal moieties. A3 Gal-T uses UDP-galactose as asource of galactose, which it transfers to an acceptor oligosaccharide,usually Gal beta (1,4)GlcNAc (N-acetyl lactosamine). As used herein theterm “alpha (1,3)galactosyltransferase” and the abbreviation “alpha1,3GT” refer to the enzyme, present in non-primate mammals, thatcatalyzes the formation of the Gal.alpha.(1,3)Gal determinant byattaching Gal in the .alpha.(1,3) position to the Gal.beta.(1,4)GlcNAcacceptor.alpha.1,3GT has the Enzyme Commission designation EC 2.4.1.124.

The expression of alpha.1-3 galactosyltransferase is regulated bothdevelopmentally and in a tissue-specific manner. The cDNA for thisenzyme has been isolated from many species, including pigs (Hoopes etal., poster presentation at the 1997 Xenotransplantation Conference,Nantes France; Katayama et al., J. Glycoconj., 15(6), 583-99 (1998);Sandrin et al., Xenotransplantation, 1, 81-88 (1994), Strahan et al.,Immunogenics, 41, 101-05 (1995)), mice (Joziasse et al., J. Biol. Chem.,267, 5534-41 (1992)), and cows (Joziasse et al., J. Biol. Chem., 264,14290-97 (1989). Some mammals do not express the Gal alpha.(1,3)Galproduct, an in these organisms the alpha 1,3GT locus is inactivated(Gailili et al., Proc. Natl. Acad. Sci. USA 15:7401, 1991). There areframeshift and nonsense substitutions within the locus, turning it intoa non-functional, processed pseudogene (Laarsen et al., J. Biol. Chem.265:7055, 1990; Joziasse et al., J. Biol. Chem. 266:6991, 1991). Larsenet al. (Proc. Natl. Acad. Sci. USA 86:8227, 1989) isolated andcharacterized a cDNA encoding murine alpha.1,3GT. Joziasse et al. (J.Biol. Chem. 267:5534, 1992) detected four distinct mRNA transcripts,which predict four different isoforms of the .alpha.1,3GT. Thefull-length mouse mRNA (including 5′ untranslated mRNA) was reported tospan at least 35-kB of genomic DNA, distributed over nine exons rangingfrom 36 base pairs to about. 2600 base pairs in length. Numbering in the5′ to 3′ direction, the coding region is distributed over Exons 4 to 9.The four transcripts are formed by alternative splicing of the pre-mRNA.Joziasse et al. (J. Biol. Chem. 264:14290, 1989) isolated andcharacterized a cDNA encoding bovine cDNA. The coding sequence waspredicted to be a membrane-bound protein with a large glycosylatedCOOH-terminal domain, a transmembrane domain, and a short NH₂ terminaldomain.

The term Gal alpha (1,3)Gal refers to an oligosaccharide determinantpresent on endothelial cells and other cells of most non-primatemammals, for which humans have a naturally occurring antibody. Exceptfor Old World monkeys, apes and humans, most mammals carry glycoproteinson their cell surfaces that contain galactose alpha 1,3-galactose(Galili et al., J. Biol. Chem. 263: 17755-17762, 1988). Humans, apes andOld World monkeys have a naturally occurring anti-alpha gal antibodythat is produced in high quantity (Cooper et al., Lancet 342:682-683,1993). It binds specifically to glycoproteins and glycolipids bearinggalactose alpha-1,3 galactose. In contrast, glycoproteins that containgalactose alpha 1,3-galactose are found in large amounts on cells ofother mammals, such as pigs. This differential distribution of the“alpha-1,3 GT epitope” and anti-Gal antibodies (i.e., antibodies bindingto glycoproteins and glycolipids bearing galactose alpha-1,3 galactose)in mammals is the result of an evolutionary process which selected forspecies with inactivated (i.e. mutated) alpha-1,3-galactosyltransferasein ancestral Old World primates and humans. Thus, humans are “naturalknockouts” of alpha-1,3GT. A direct outcome of this event is therejection of xenografts, such as the rejection of pig organstransplanted into humans initially via HAR.

Alpha 1,3 N-Acetylgalactosaminyltransferase (a3 GalNAc-T)

The present invention features the structure-based design of alpha 1-3N-Acetylgalactosaminyltransferase (a3 GalNAc-T) from a3Gal-T that cantransfer 2′-modified galactose from the corresponding UDP-derivatives.The genetically engineered a3Gal-T to a3GalNAc-T, which can transfer2′N-acetygalactose (GalNAc) or 2′-modified galactose from thecorresponding UDP-derivatives, is very useful for the synthesis of atrisaccharide GalNAccd-3Galβ1-4Glc or GalNAccd-3-Gal β1-4GlcNAc or2′-modified-Galα1-3Galβ1-4Glc or 2′-modified-Galα1-3-Galβ1-4GlcNAc in anoligosaccharide chain that is otherwise difficult to be synthesized bychemical methods.

The Sugar Binding Pocket

It has been discovered that mutation of alpha 1,3N-Acetylgalactosaminyltransferase (a3 GalNAc-T) can broaden the donorspecificity of the enzyme. More specifically, it has been determinedthat, in certain embodiments, mutation in residues in thesugar-nucleotide binding pocket of the enzyme can broaden the donorspecificity of the enzyme. In particular, substitution of amino acidresidues located in the in the sugar-nucleotide binding pocket providegreater flexibility and decreased steric hindrance that allow a broaderrange of donor (or substrate) binding, for example UDP-GalNAc,UDP-galNAc analogues, UDP-galactose, or UDP-galactose analogues, whilestill preserving interaction with amino acid residues active duringcatalytic bond formation between the donor and the acceptor.

A three-residue motif, Asp-X-Asp (DXD) or Glu-X-Asp (EXD), or itsequivalent generally participates in metal ion binding in enzymes of theGT-A fold. Enzymes of the GT-B fold such as the microbialglycosyltransferases MurG (Hu, Y. et al. (2003)) and GtfB (Mulichack etal. 2001), and BGT (Morera et al. 1999), do not have a DXD motif or itsequivalent, even though some, BGT for example, require a metal ion foractivity. In glycosyltransferases that require Mn2C ion as cofactor, themetal ion is bound in an octahedral coordination (Qasba et al. 2005). Itinteracts with one or both acidic residues of the DXD or EXD motif andwith two oxygen atoms from the a-phosphate and b-phosphate

of UDP. To satisfy the octahedral geometry, the three remaining metalion links are made either to water molecules or to water in combinationwith other residues of the protein. In several glycosyltransferases onlythe first (Lobsanov, Y. D. et al. (2004)) or the second (Gastinel et al.1999; Ramakrishnan et al. 2001; Ramakrishnan 2002; Unligil 2000) acidicresidue of the motif coordinates directly with the metal ion. Forexample, in some enzymes, the first acidic residue of the motif eitherinteracts directly with the sugar donor or the ribose moiety orinteracts via the water molecules coordinated to the Mn2C ion. In bloodgroup A and B and alpha 3GT transferases, by contrast, both asparticacid residues of the DXD motif directly coordinate the metal ion.

The crystal structures of several glycosyltransferases of either theGT-A or GT-B fold show that at least one flexible loop region has acrucial role in the catalytic mechanism of the enzyme (Qasba et al.2005). Although the exact location of this loop differs among thetransferases, it is invariably located in the vicinity of thesugarnucleotide-binding site. Owing to the flexibility of this region,the loop structure cannot be traced in the apo form of the enzyme, whichlacks bound substrate. In the sugar-nucleotide-bound structures, theloop either is in a closed conformation covering the bound donorsubstrate or is found disordered in the vicinity of thesugarnucleotide-binding site. In a3GT, the C-terminal 11-residueflexible loop changes its conformation when the sugamucleotide donor isbound (Boix et al., 2001).

Without being bound by any theory, examples of catalytic residuesthought to be important for binding include Pro 191, Gln228, His280,A1a28 1, and A1a282 of bovine a3 GalT. Accordingly, the inventionprovides alpha 1,3 N-Acetylgalactosaminyltransferase (a3 GalNAc-T)enzymess having amino acid substitutions, insertions, and deletions thatprovide greater flexibility and decreased steric hindrance in the sugarnucleotide binding pocket to allow the mutated alpha 1,3N-Acetylgalactosaminyltransferase (a3 GalNAc-T) to catalyze chemicalbonding of the donor to an acceptor, such as N-acetylglucosamine(GlcNAc), galactose (Gal) and xylose residues of glycoproteins,glycolipids or proteoglycan (glycoconjugates).

Polypeptide Fragments

The invention features, in certain embodiments, polypeptide fragmentsfrom alpha 1,3 N-Acetylgalactosaminyltransferases (alpha3GalNac-T)transfer sugars with a chemically reactive functional group from a sugardonor to a sugar acceptor. The a3 GalNAc transferases described hereincomprise substitutions in a3 Gal-T, in certain preferred examples,bovein a 3GalT. The substitutions have the effect of broadening thedonor specificity of the transferase and make the enzyme an alpha 3GalNAc-T.

In certain examples, the invention provides a polypeptide fragment of analpha 1,3 N-Acetylgalactosylaminotransferase (alpha 3GalNaC T) thatretains the ability to transfer a sugar with a chemically reactivefunctional group from a sugar donor to a sugar acceptor. The polypeptidefragments, in certain preferred examples, comprise a donorsubstrate-binding site, a hinge region and a DXD motif. Thr polypeptidefragment can comprise one or more substitutions in the donorsubstrate-binding site.

A number of substitutions of the polypeptide fragments are envisioned bythe instant invention. The substitutions have the effect of broadeningthe donor specificity of the enzyme. In certain examples, thepolypeptide fragment comprises one or more substitutions in the hingeregion. In other examples, polypeptide fragment comprise one or moresubstitutions near the DXD motif.

the one or more substitutions in the substrate binding site comprise anamino acid substitution at position 280, 281, or 282 corresponding tobovine alpha 1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21).In another related embodiment, the one or more substitutions in thesubstrate hinge region comprise an amino acid substitution at position191 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO: 21). In one embodiment, the one or more substitutionsclose to the DXD motif comprise an amino acid substitution at position228 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO 21). In another embodiment, wherein a serine (S) issubstituted for a histidine (H) at amino acid position 280, a glycine(G) is substituted for an alanine (A) at amino acid position 281, or aglycine (G) is substituted for an alanine at amino acid position 282 of(SEQ ID NO 21). In another embodiment, a serine (S) or an alanine (A) issubstituted for a proline (P) at amino acid position 191 correspondingto (SEQ ID NO: 21). In still another embodiment, a glutamine (Q) isreplaced with a a methionine (M) at amino acid position 228 of (SEQ IDNO:21).

In another example, the invention features a polypeptide fragment of analpha 1,3 N-Acetylgalactosaminyltransferase (alpha3GalNac-T) thattransfers a sugar with a chemically reactive functional group from asugar donor to a sugar acceptor, wherein the polypeptide fragmentcomprises and one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, or SEQ ID NO: 20. The sequences are set forth below.

In certain preferred embodiments, the one or more substitutions comprisean amino acid exchange at position 191. SEQ ID NO: 1 and SEQ ID NO: 2correspond to the nucleotide and amino acid sequences, respectively,encoding the P191A bovine alpha3GalT.

SEQ ID NO: 1   1 ATGGCTAGCA TGACTGGNGN NCAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTTGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGGCTGAGA 401AGAGGTGGCA GGATATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGATGT 501GGACCAGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTTATTAC CATGCAGCCA TTTTTGGGGG AACACCCACT CAGGTCCTTA 701ACATCACCCA GGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCC ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTCGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGTC SEQ ID NO: 2   1MASMTGXQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVW  51EGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLT SANKHFMVGH 101PVIFYIMVDD VSRMPLIELG PLRSFKVFKI KAEKRWQDIS   MMRMKTIGEH 151IVAHIQHEVD FLFCMDVDQV FQDKFGVETL GESVAQLQAW WYKADPNDFT 201YERRKESAAY IPFGEGDFYY HAAIFGGTPT QVLNITQECF KGILKDKKND 251IEAQWHDESH LNKYFLLNKP TKILSPEYCW DYHIGLPSDI KLVKMSWQTK 301 EYNVVRNNV

In certain preferred embodiments, the one or more substitutions comprisean amino acid exchange at position 280, 281 or 282. SEQ ID NO: 3 and SEQID NO: 4 correspond to the nucleotide and amino acid sequences,respectively, encoding the H280L A281G A282G bovine alpha3GalT.

SEQ ID NO: 3   1 ATGGCTAGCA TGACTGGTGG NCAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTTGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGCCTGAGA 401AGAGGTGGCA GGACATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGATGT 501GGACCAGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTTATTAC CTAGGAGGCA TTTTTGGGGG AACACCCACT CAGGTCCTTA 701ACATCACCCA GGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCC ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTCGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGTC SEQ ID NO: 4   1MASMTGGQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVW  51EGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLT SANKHFMVGH 101PVIFYIMVDD VSRMPLIELG PLRSFKVFKI KPEKRWQDIS MMRMKTIGEH 151IVAHIQHEVD FLFCMDVDQV FQDKFGVETL GESVAQLQAW WYKADPNDFT 201YERRKESAAY IPFGEGDFYY LGGIFGGTPT QVLNITQECF KGILKDKKND 251IEAQWHDESH LNKYFLLNKP TKILSPEYCW DYHIGLPSDI KLVKMSWQTK 301 EYNVVRNNV

In certain preferred embodiments, the one or more substitutions comprisean amino acid exchange at position 228, 280, 281, or 282. SEQ ID NO: 5and SEQ ID NO: 6 correspond to the nucleotide and amino acid sequences,respectively, encoding the Q228M H280S A281G A282G bovine alpha3GalT.

SEQ ID NO: 5   1 ATGGCTAGCA TGACTGGTGG NCAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTTGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGGCTGAGA 401AGAGGTGGCA GGATATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGACGT 501CGACATGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTTATTAC TCCGGAGGCA TTTTTGGGGG AACACCCACT CAGGTCCTTA 701ACATCACCCA GGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCT ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTTGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGTC SEQ ID NO: 6   1MASMTGGQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVW  51EGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLT SANKHFMVGH 101PVIFYIMVDD VSRMPLIELG PLRSFKVFKI KAEKRWQDIS MMRMKTIGEH 151IVAHIQHEVD FLFCMDVDMV FQDKFGVETL GESVAQLQAW WYKADPNDFT 201YERRKESAAY IPFGEGDFYY SGGIFGGTPT QVLNITQECF KGILKDKKND 251IEAQWHDESH LNKYFLLNKP TKILSPEYCW DYHIGLPSDI KLVKMSWQTK 301 EYNVVRNNV

In certain preferred embodiments, the one or more substitutions comprisean amino acid exchange at position 280 or 282. SEQ ID NO: 7 and SEQ IDNO: 8 correspond to the nucleotide and amino acid sequences,respectively, encoding the H280S A282G bovine alpha3GalT.

SEQ ID NO: 7   1 ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTGGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGCCTGAGA 401AGAGGTGGCA GGACATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGATGT 501GGACCAGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTTATTAC TCCGCCGGCA TTTTTGGGGG AACACCCACT CAGGTCCTTA 701ACATCACCCA GGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCT ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTTGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGTCTGA SEQ ID NO: 8   1MASMTGGQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVW  51EGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLT SANKHFMVGH 101PVIFYIMVDD VSRMPLIELG PLRSFKVFKI KPEKRWQDIS MMRMKTIGEH 151IVAHIQHEVD FLFCMDVDQV FQDKFGVETL GESVAQLQAW WYKADPNDFT 201YERRKESAAY IPFGEGDFYY SAGIFGGTPT QVLNITQECF KGILKDKKND 251IEAQWHDESH LNKYFLLNKP TKILSPEYCW DYHIGLPSDI KLVKMSWQTK 301 EYNVVRNNV*

In certain preferred embodiments, the one or more substitutions comprisean amino acid exchange at position 191, 280, 281, or 282. SEQ ID NO: 9and SEQ ID NO: 10 correspond to the nucleotide and amino acid sequences,respectively, encoding the P191S H280S A281G A282G bovine alpha3GalT.

SEQ ID NO: 9   1 ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTTGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGTCTGAGA 401AGAGGTGGCA GGATATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGATGT 501GGACCAGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTTATTAC TCCGGAGGCA TTTTTGGGGG AACACCCACT CAGGTCCTTA 701ACATCACCCA GGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCC ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTCGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGTC SEQ ID NO: 10   1MASMTGGQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVW  51EGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLT SANKHFMVGH 101PVIFYIMVDD VSRMPLIELG PLRSFKVFKI KSEKRWQDIS MMRMKTIGEH 151IVAHIQHEVD FLFCMDVDQV FQDKFGVETL GESVAQLQAW WYKADPNDFT 201YERRKESAAY IPFGEGDFYY SGGIFGGTPT QVLNITQECF KGILKDKKND 251IEAQWHDESH LNKYFLLNKP TKILSPEYCW DYHIGLPSDI KLVKMSWQTK 301 EYNVVRNNV

In certain preferred embodiments, the one or more substitutions comprisean amino acid exchange at position 191, 280, 281, or 282. SEQ ID NO: 11and SEQ ID NO: 12 correspond to the nucleotide and amino acid sequences,respectively, encoding the P191A H280S A281G A282G bovine alpha3GalT.

SEQ ID NO: 11   1 ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTTGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGGCTGAGA 401AGAGGTGGCA GGATATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGATGT 501GGACCAGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTTATTAC TCCGGAGGCA TTTTTGGGGG AACACCCACT CANNTCCTTA 701ACATCACCCA NGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCC ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTCGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGT SEQ ID NO: 12   1MASMTGGQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVW  51EGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLT SANKHFMVGH 101PVIFYIMVDD VSRMPLIELG PLRSFKVFKI KAEKRWQDIS MMRMKTIGEH 151IVAHIQHEVD FLFCMDVDQV FQDKFGVETL GESVAQLQAW WYKADPNDFT 201YERRKESAAY IPFGEGDFYY SGGIFGGTPT XXLNITXECF KGILKDKKND 251IEAQWHDESH LNKYFLLNKP TKILSPEYCW DYHIGLPSDI KLVKMSWQTK 301 EYNVVRNN

In certain preferred embodiments, the one or more substitutions comprisean amino acid exchange at position 278, 280, 281, or 282. SEQ ID NO: 13and SEQ ID NO: 14 correspond to the nucleotide and amino acid sequences,respectively, encoding the Y278L H280S A281G A282G bovine alpha3GalT.

SEQ ID NO: 13   1 ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTTGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGCCTGAGA 401AGAGGTGGCA GGACATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGATGT 501GGACCAGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTCTTTAC TCCGGAGGCA TTTTTGGGGG AACACCCACT CAGGTCCTTA 701ACATCACCCA GGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCC ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTCGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGTCTGA SEQ ID NO: 14   1MASMTGGQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVW  51EGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLT SANKHFMVGH 101PVIFYIMVDD VSRMPLIELG PLRSFKVFKI KPEKRWQDIS MMRMKTIGEH 151IVAHIQHEVD FLFCMDVDQV FQDKFGVETL GESVAQLQAW WYKADPNDFT 201YERRKESAAY IPFGEGDFLY SGGIFGGTPT QVLNITQECF KGILKDKKND 251IEAQWHDESH LNKYFLLNKP TKILSPEYCW DYHIGLPSDI KLVKMSWQTK 301 EYNVVRNNV*

In certain preferred embodiments, the one or more substitutions comprisean amino acid exchange at position 191. SEQ ID NO: 15 and SEQ ID NO: 16correspond to the nucleotide and amino acid sequences, respectively,encoding the P191S bovine alpha3GalT.

SEQ ID NO: 15   1 ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTTGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGTCTGAGA 401AGAGGTGGCA GGATATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGATGT 501GGACCAGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTTATTAC CATGCAGCCA TTTTTGGGGG AACACCCACT CAGGTCCTTA 701ACATCACCCA GGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCC ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTCGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGTC SEQ ID NO: 16MASMTGGQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVWEGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLTSANKHFMVGH PVIFYIMVDD VSRMPLIELG PLRSFKVFKIKSEKRWQDIS MMRMKTIGEH IVAHIQHEVD FLFCMDVDQVFQDKFGVETL GESVAQLQAW WYKADPNDFTYERRKESAAY IPFGEGDFYY HAAIFGGTPT QVLNITQECFKGILKDKKND IEAQWHDESH LNKYFLLNKP TKILSPEYCWDYHIGLPSDI KLVKMSWQTK EYNVVRNNV

In certain preferred embodiments, the one or more substitutions areamino acid exchange at position 280, 281 or 282. SEQ ID NO: 17 and SEQID NO: 18 correspond to the nucleotide and amino acid sequences,respectively, encoding the H280S A281G A282G bovine alpha3GalT.

SEQ ID NO: 17   1 ATGGCTAGCA TGACTGGTGG NCAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTTGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGCCTGAGA 401AGAGGTGGCA GGACATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGATGT 501GGACCAGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTTATTAC TCCGGAGGCA TTTTTGGGGG AACACCCACT CAGGTCCTTA 701ACATCACCCA GGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCC ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTCGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGTC SEQ ID NO: 18   1MASMTGGQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVW  51EGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLT SANKHFMVGH 101PVIFYIMVDD VSRMPLIELG PLRSFKVFKI KPEKRWQDIS MMRMKTIGEH 151IVAHIQHEVD FLFCMDVDQV FQDKFGVETL GESVAQLQAW WYKADPNDFT 201YERRKESAAY IPFGEGDFYY SGGIFGGTPT QVLNITQECF KGILKDKKND 251IEAQWHDESH LNKYFLLNKP TKILSPEYCW DYHIGLPSDI KLVKMSWQTK 301 EYNVVRNNV

In certain preferred embodiments, the one or more substitutions comprisean amino acid exchange at position 280, 281 or 282. SEQ ID NO: 19 andSEQ ID NO: 20 correspond to the nucleotide and amino acid sequences,respectively, encoding the H280T A281G A282G bovine alpha3GalT.

SEQ ID NO: 19   1 ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGCGGAT CCCACCACCA 51 CCACCACCAC GAAAGCAAGC TTAAGCTATC GGACTGGTTC AACCCATTTA 101AACGCCCCGA GGTTGTGACC ATGACGAAGT GGAAGGCTCC AGTGGTGTGG 151GAAGGCACTT ACAACAGAGC CGTCTTAGAC AATTATTATG CCAAGCAGAA 201AATTACCGTC GGCCTGACGG TTTTCGCCGT CGGAAGATAC ATTGAGCATT 251ACTTGGAGGA GTTCTTAACG TCTGCTAATA AGCACTTCAT GGTGGGCCAC 301CCAGTCATCT TTTATATCAT GGTAGATGAT GTCTCCAGGA TGCCTTTGAT 351AGAGTTGGGT CCTCTGCGCT CCTTCAAAGT GTTTAAGATC AAGCCTGAGA 401AGAGGTGGCA GGACATCAGC ATGATGCGCA TGAAGACTAT CGGGGAGCAC 451ATTGTGGCCC ACATCCAGCA TGAGGTTGAC TTCCTTTTCT GCATGGATGT 501GGACCAGGTC TTCCAAGACA AGTTTGGGGT GGAGACCCTG GGCGAGTCGG 551TGGCCCAGCT ACAAGCCTGG TGGTACAAGG CAGATCCCAA TGACTTCACC 601TACGAGAGGC GGAAGGAGTC TGCAGCATAC ATTCCCTTCG GCGAAGGGGA 651TTTTTATTAC ACAGGAGGTA TTTTTGGGGG AACACCCACT CAGGTCCTTA 701ACATCACCCA GGAATGCTTC AAAGGAATCC TCAAGGACAA GAAAAATGAC 751ATAGAAGCCC AATGGCATGA TGAAAGCCAT CTAAACAAGT ATTTCCTTCT 801CAACAAACCT ACTAAAATCT TATCCCCGGA ATACTGCTGG GATTATCACA 851TAGGCCTACC TTCGGATATT AAGCTTGTCA AGATGTCTTG GCAGACAAAA 901GAGTATAATG TGGTTAGAAA TAATGTC SEQ ID NO: 20   1MASMTGGQQM GRGSHHHHHH ESKLKLSDWF NPFKRPEVVT MTKWKAPVVW  51EGTYNRAVLD NYYAKQKITV GLTVFAVGRY IEHYLEEFLT SANKHFMVGH 101PVIFYIMVDD VSRMPLIELG PLRSFKVFKI KPEKRWQDIS MMRMKTIGEH 151IVAHIQHEVD FLFCMDVDQV FQDKFGVETL GESVAQLQAW WYKADPNDFT 201YERRKESAAY IPFGEGDFYY TGGIFGGTPT QVLNITQECF KGILKDKKND 251IEAQWHDESH LNKYFLLNKP TKILSPEYCW DYHIGLPSDI KLVKMSWQTK 301 EYNVVRNNV

In certain preferred embodiments, SEQ ID NO: 21 corresponds to the aminoacid sequence encoding the full length bovine alpha3GalT.

SEQ ID NO: 21   1 MNVKGKVILS MLVVSTVIVV FWEYIHSPEG SLFWINPSRNPEVGGSSIQK GWWLPRWFNN  61 GYHEEDGDIN EEKEQRNEDE SKLKLSDWFN PFKRPEVVTMTKWKAPVVWE GTYNRAVLDN 121 YYAKQKITVG LTVFAVGRYI EHYLEEFLTS ANKHFMVGHPVIFYIMVDDV SRMPLIELGP 181 LRSFKVFKIK PEKRWQDISM MRMKTIGEHI VAHIQHEVDFLFCMDVDQVF QDKFGVETLG 241 ESVAQLQAWW YKADPNDFTY ERRKESAAYI PFGEGDFYYHAAIFGGTPTQ VLNITQECFK 301 GILKDKKNDI EAQWHDESHL NKYFLLNKPT KILSPEYCWDYHIGLPSDIK LVKMSWQTKE 361 YNVVRNNV

An isolated gene includes a gene which is essentially free of sequenceswhich naturally flank the gene in the chromosomal DNA of the organismfrom which the gene is derived (i.e., is free of adjacent codingsequences which encode a second or distinct polypeptide or RNA molecule,adjacent structural sequences or the like) and optionally includes 5′and 3′ regulatory sequences, for example promoter sequences and/orterminator sequences.

The invention features, in certain embodiments, isolated nucleic acidmolecules. The isolated nucleic acid molecules may, in certain examples,comprise a nucleotide sequence which is at least 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or more homologous to the nucleotide sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO; 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ IDNO: 19 or a complement thereof.

A nucleic acid molecule of the present invention (e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO; 11, SEQ ID NO: 13,SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19 or a complement thereof)can be isolated using standard molecular biology techniques and thesequence information provided herein. For example, nucleic acidmolecules can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook, J., Fritsh, E. F., andManiatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) or can be isolated by the polymerase chainreaction using synthetic oligonucleotide primers designed based upon thesequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO; 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, orSEQ ID NO: 19 or a complement thereof. A nucleic acid of the inventioncan be amplified using cDNA, mRNA or alternatively, genomic DNA, as atemplate and appropriate oligonucleotide primers according to standardPCR amplification techniques. An isolated nucleic acid molecule of theinvention can, in certain examples, comprise a nucleic acid moleculewhich is a complement of the nucleotide sequence shown in SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO; 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

In certain embodiments, the isolated nucleic acid molecule can encode apolypeptide that comprises an amino acid sequence that is at least about50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous tothe amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO; 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, or SEQ ID NO: 20.

Nucleic Acids and Vectors

As described herein, the present invention provides isolated nucleicacid segments that encode polypeptide fragments of alpha1-3N-Acetylgalactosaminyltransferase (alpha 3 GalNAc-T). In certainembodiments, for example, the substitutions are in bovine a3Gal-T.

In certain examples, the one or more substitutions in the substratebinding site comprise an amino acid substitution at position 280, 281,or 282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or moresubstitutions in the substrate hinge region comprise an amino acidsubstitution at position 191 corresponding to bovine alpha 1,3galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In oneembodiment, the one or more substitutions close to the DXD motifcomprise an amino acid substitution at position 228 corresponding tobovine alpha 1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21).In another embodiment, wherein a serine (S) is substituted for ahistidine (H) at amino acid position 280, a glycine (G) is substitutedfor an alanine (A) at amino acid position 281, or a glycine (G) issubstituted for an alanine at amino acid position 282 of (SEQ ID NO 21).In another embodiment, a serine (S) or an alanine (A) is substituted fora proline (P) at amino acid position 191 corresponding to (SEQ ID NO:21). In still another embodiment, a glutamine (Q) is replaced with amethionine (M) at amino acid position 228 of (SEQ ID NO: 21).

Nucleic acid sequences encoding alpha 3 GalNAc-T enzymes, for exampleSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO; 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ IDNO: 19, as well as other alpha GalNAc-T from other organisms areavailable. These nucleic acid sequences can be modified to encode thepolypeptide fragments and amino acid segments of the invention throughuse of well-known techniques (Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (2001)). For example, a portion of the nucleic acidsequence encoding alpha 1,3 GalNAc-T, for example SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO; 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, can be insertedinto an expression vector such that an amino acid segment correspondingto the polypeptide fragment of any of the alpha GalNAc-T enzymes thattransfers a sugar with a chemically reactive functional group from asugar donor to a sugar acceptor (for example, but not limited to, SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO; 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19)is expressed upon transformation of a cell with the expression vector.The nucleic acid segments of the invention may be optimized forexpression in select cells. Codon optimization tables are available.Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1988.

The nucleic acid segments can be inserted into numerous types ofvectors. A vector may include, but is not limited to, any plasmid,phagemid, F-factor, virus, cosmid, or phage in double or single strandedlinear or circular form, which may or may not be self-transmissible ormobilizable. The vector can also transform a prokaryotic or eukaryotichost either by integration into the cellular genome or existextrachromosomally (e.g. autonomous replicating plasmid with an originof replication).

Preferably the nucleic acid segment in the vector is under the controlof, and operably linked to, an appropriate promoter or other regulatoryelements for transcription in vitro or in a host cell such as aeukaryotic cell or microbe, e.g. bacteria. The vector may be abi-functional expression vector which functions in multiple hosts. Inthe case of genomic DNA, this may contain its own promoter or otherregulatory elements and in the case of cDNA this may be under thecontrol of a promoter or other regulatory sequences for expression in ahost cell.

Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from bacteria and eukaryotic cells(e.g. mammalian, yeast or fungal).

The vector may also be a cloning vector which typically contains one ora small number of restriction endonuclease recognition sites at whichnucleic acid segments can be inserted in a determinable fashion. Suchinsertion can occur without loss of essential biological function of thecloning vector. A cloning vector may also contain a marker gene that issuitable for use in the identification and selection of cellstransformed with the cloning vector. Examples of marker genes aretetracycline resistance, hygromycin resistance or ampicillin resistance.Many cloning vectors are commercially available (Stratagene, New EnglandBiolabs, Clonetech).

The nucleic acid segments of the invention may also be inserted into anexpression vector. Typically an expression vector contains (1)prokaryotic DNA elements coding for a bacterial replication origin andan antibiotic resistance gene to provide for the amplification andselection of the expression vector in a bacterial host; (2) regulatoryelements that control initiation of transcription such as a promoter;and (3) DNA elements that control the processing of transcripts such asintrons, transcription termination/polyadenylation sequence.

Methods to introduce a nucleic acid segment into a vector are well knownin the art (Sambrook et al., 1989). Briefly, a vector into which thenucleic acid segment is to be inserted is treated with one or morerestriction enzymes (restriction endonuclease) to produce a linearizedvector having a blunt end, a “sticky” end with a 5′ or a 3′ overhang, orany combination of the above. The vector may also be treated with arestriction enzyme and subsequently treated with another modifyingenzyme, such as a polymerase, an exonuclease, a phosphatase or a kinase,to create a linearized vector that has characteristics useful forligation of a nucleic acid segment into the vector. The nucleic acidsegment that is to be inserted into the vector is treated with one ormore restriction enzymes to create a linearized segment having a bluntend, a “sticky” end with a 5′ or a 3′ overhang, or any combination ofthe above. The nucleic acid segment may also be treated with arestriction enzyme and subsequently treated with another DNA modifyingenzyme. Such DNA modifying enzymes include, but are not limited to,polymerase, exonuclease, phosphatase or a kinase, to create apolynucleic acid segment that has characteristics useful for ligation ofa nucleic acid segment into the vector.

The treated vector and nucleic acid segment are then ligated together toform a construct containing a nucleic acid segment according to methodsknown in the art (Sambrook, 2002). Briefly, the treated nucleic acidfragment and the treated vector are combined in the presence of asuitable buffer and ligase. The mixture is then incubated underappropriate conditions to allow the ligase to ligate the nucleic acidfragment into the vector. It is preferred that the nucleic acid fragmentand the vector each have complimentary “sticky” ends to increaseligation efficiency, as opposed to blunt-end ligation. It is morepreferred that the vector and nucleic acid fragment are each treatedwith two different restriction enzymes to produce two differentcomplimentary “sticky” ends. This allows for directional ligation of thenucleic acid fragment into the vector, increases ligation efficiency andavoids ligation of the ends of the vector to reform the vector withoutthe inserted nucleic acid fragment.

Suitable prokaryotic vectors include but are not limited to pBR322,pMB9, pUC, lambda bacteriophage, m13 bacteriophage, and Bluescript®Suitable eukaryotic vectors include but are not limited to PMSG,pAV009/A+, PMT010/A+, pMAM neo-5, bacculovirus, pDSVE, YIPS, YRP17, YEP.It will be clear to one of ordinary skill in the art which vector orpromoter system should be used depending on which cell type is used fora host cell.

The invention also provides expression cassettes which contain a controlsequence capable of directing expression of a particular nucleic acidsegment of the invention either in vitro or in a host cell. Theexpression cassette is an isolatable unit such that the expressioncassette may be in linear form and functional in in vitro transcriptionand translation assays. The materials and procedures to conduct theseassays are commercially available from Promega Corp. (Madison, Wis.).For example, an in vitro transcript may be produced by placing a nucleicacid segment under the control of a T7 promoter and then using T7 RNApolymerase to produce an in vitro transcript. This transcript may thenbe translated in vitro through use of a rabbit reticulocyte lysate.Alternatively, the expression cassette can be incorporated into a vectorallowing for replication and amplification of the expression cassettewithin a host cell or also in vitro transcription and translation of anucleic acid segment.

Such an expression cassette may contain one or a plurality ofrestriction sites allowing for placement of the nucleic acid segmentunder the regulation of a regulatory sequence. The expression cassettecan also contain a termination signal operably linked to the nucleicacid segment as well as regulatory sequences required for propertranslation of the nucleic acid segment. Expression of the nucleic acidsegment in the expression cassette may be under the control of aconstitutive promoter or an inducible promoter, which initiatestranscription only when the host cell is exposed to some particularexternal stimulus.

The expression cassette may include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, anucleic acid segment and a transcriptional and translational terminationregion functional in vivo and/or in vitro. The termination region may benative with the transcriptional initiation region, may be native withthe nucleic acid segment, or may be derived from another source.Numerous termination regions are known in the art. Guerineau et al.,Mol. Gen. Genet., 262:141 (1991); Proudfoot, Cell, 64:671 (1991);Sanfacon et al., Genes Dev., 5:141 (1991); Munroe et al., Gene, 91:151(1990); Ballas et al., Nucleic Acids Res., 17:7891 (1989); Joshi et al.,Nucleic Acid Res., 15:9627 (1987).

The regulatory sequence can be a nucleic acid sequence located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influences the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences can include, but are not limited to,enhancers, promoter and repressor binding sites, translation leadersequences, introns, and polyadenylation signal sequences. They mayinclude natural and synthetic sequences as well as sequences that may bea combination of synthetic and natural sequences. While regulatorysequences are not limited to promoters, some useful regulatory sequencesinclude constitutive promoters, inducible promoters, regulatedpromoters, tissue-specific promoters, viral promoters and syntheticpromoters.

A promoter is a nucleotide sequence that controls expression of thecoding sequence by providing the recognition for RNA polymerase andother factors required for proper transcription. A promoter includes aminimal promoter, consisting only of all basal elements needed fortranscription initiation, such as a TATA-box and/or initiator that is ashort DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. A promoter maybe inducible. Several inducible promoters have been reported (CurrentOpinion in Biotechnology, 7:168 (1996)). Examples include thetetracycline repressor system, Lac repressor system, copper-induciblesystems, salicylate-inducible systems (such as the PR1a system). Alsoincluded are the benzene sulphonamide—(U.S. Pat. No. 5,364,780,incorporated by reference herein) and alcohol—(WO 97/06269 and WO97/06268, both incorporated by reference herein) inducible systems andglutathione S-transferase promoters. In the case of a multicellularorganism, the promoter can also be specific to a particular tissue ororgan or stage of development.

An enhancer is a DNA sequence which can stimulate promoter activity andmay be an innate element of the promoter or a heterologous elementinserted to enhance the level or tissue specificity of a promoter. It iscapable of operating in both orientations (normal or flipped), and iscapable of functioning even when moved either upstream or downstreamfrom the promoter. Both enhancers and other upstream promoter elementsbind sequence-specific DNA-binding proteins that mediate their effects.

The expression cassette can contain a 5′ non-coding sequence which is anucleotide sequence located 5′ (upstream) to the coding sequence. It ispresent in the fully processed mRNA upstream of the initiation codon andmay affect processing of the primary transcript to mRNA, stability ofthe mRNA, or translation efficiency (Turner et al., MolecularBiotechnology, 3:225 (1995)).

The expression cassette may also contain a 3′ non-coding sequence, whichis a nucleotide sequence, located 3′ (downstream) to a coding sequenceand includes polyadenylation signal sequences and other sequencesencoding regulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor.

The invention also provides a construct containing a vector and anexpression cassette. The vector may be selected from, but not limitedto, any vector previously described. Into this vector may be inserted anexpression cassette through methods known in the art and previouslydescribed (Sambrook et al., 1989). In one embodiment, the regulatorysequences of the expression cassette may be derived from a source otherthan the vector into which the expression cassette is inserted. Inanother embodiment, a construct containing a vector and an expressioncassette is formed upon insertion of a nucleic acid segment of theinvention into a vector that itself contains regulatory sequences. Thus,an expression cassette is formed upon insertion of the nucleic acidsegment into the vector. Vectors containing regulatory sequences areavailable commercially and methods for their use are known in the art(Clonetech, Promega, Stratagene).

The expression cassette, or a vector construct containing the expressioncassette may be inserted into a cell. The expression cassette or vectorconstruct may be carried episomal or integrated into the genome of thecell.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a cellular host. Transformationof bacteria and many eukaryotic cells may be accomplished through use ofpolyethylene glycol, calcium chloride, viral infection, phage infection,electroporation and other methods known in the art. Other transformationmethods are available to those skilled in the art, such as direct uptakeof foreign DNA constructs (see EP 295959, incorporated by referenceherein), techniques of electroporation or high velocity ballisticbombardment with metal particles coated with the nucleic acid constructs(U.S. Pat. No. 4,945,050, incorporated by reference herein).

The selection of an appropriate expression vector will depend upon themethod of introducing the expression vector into host cells. Typicallyan expression vector contains (1) prokaryotic DNA elements coding for abacterial origin of replication and an antibiotic resistance gene toprovide for the amplification and selection of the expression vector ina bacterial host; (2) DNA elements that control initiation oftranscription, such as a promoter; (3) DNA elements that control theprocessing of transcripts, such as introns, transcriptiontermination/polyadenylation sequence; and (4) a reporter gene that isoperatively linked to the DNA elements to control transcriptioninitiation. Useful reporter genes include .beta.-galactosidase,chloramphenicol acetyl transferase, luciferase, green fluorescentprotein (GFP) and the like.Methods of Making and Folding

Galactosyltransferase enzymes of the invention may be produced insoluble form. Methods that may be used to produce such soluble enzymeshave been described (U.S. Pat. No. 5,032,519, incorporated by referencein its entirety herein). Briefly, a hydrophobic transmembrane anchorregion of a galactosyltransferase is removed to produce an enzyme thatis in soluble form.

Accordingly, the invention features methods of making an oligosaccharidecomprising incubating a reaction mixture comprising a polypeptidefragment of an alpha 1,3 N-Acetylgalactosylaminotransferase (alpha3GalNaC T) that retains the ability to transfer a sugar with achemically reactive functional group with a sugar donor and a sugaracceptor. In certain examples, the polypeptide fragment can comprise anyone of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, orSEQ ID NO: 20.

Alternatively, alpha 1,3 GalNAcT enzymes of the invention may beproduced such that they are anchored in the membrane of a cell. Suchenzymes may be produced that are anchored in the membranes ofprokaryotic and eukaryotic cells. Methods to produce such enzymes havebeen described (U.S. Pat. No. 6,284,493, incorporated by reference inits entirety herein).

Briefly, in the case of procaryotes, the signal and transmembranesequences of the transferase, for example the alpha 1,3 GalNAcT enzymeof the invention, are replaced by a bacterial signal sequence, capableof effecting localization of the fusion protein to the outer membrane.Suitable signal sequences include, but are not limited to those from themajor E. coli lipoprotein Lpp and lam B. In addition, membrane spanningregions from Omp A, Omp C, Omp F or Pho E can be used in a tripartitefusion protein to direct proper insertion of the fusion protein into theouter membrane. Any procaryotic cells can be used in accordance with thepresent invention including but not limited to E. coli, Bacillus sp.,and Pseudomonas sp. as representative examples.

It is also possible, in certain embodiments, that the nativetransmembrane domain of the glycosyltransferase, for example theengineered GalNAcT of the invention as described herein, is replaced bythe transmembrane domain of a bacterial outer membrane protein. In thisembodiment, the alpha 1,3 GalNAcT signal sequence and the bacterialtransmembrane region act in concert to anchor the galactosyltransferaseto the bacterial outer cell membrane. Nearly any outer membrane boundprotein is suitable for this use including but not limited to Omp A, OmpC, and Omp F, Lpp, and Lam B. The catalytic portion of the GalNAcTshould be fused to an extracellular loop in the bacterial transmembraneregion in order to insure proper orientation of the fusion protein onthe outer membrane surface and not in the cytoplasm or periplasm of thecell. Insertion of a protein into such a loop region has been previouslyreported (Charbit et al., J. Bacteriology, 173:262 (1991); Francisco etal., Proc. Natl. Acad. Sci., 89:2713 (1992)).

The present invention is also applicable for use with eukaryotic cellsresulting in cell surface expression of glycosyltransferases in knownculturable eucaryotic cells including but not limited to yeast cells,insect cells, chinese hamster ovary cells (CHO cells), mouse L cells,mouse A9 cells, baby hamster kidney cells, C127 cells, COS cells, Sf9cells, and PC8 cells.

In another embodiment of the present invention, the transmembrane domainof the alpha 1,3 GalNAcT can be replaced by the transmembrane domain ofa plasma membrane protein. The transmembrane domain of any residentplasma membrane protein will be appropriate for this purpose. Thetransmembrane portions of the M6 P/IGF-II receptor, LDL receptor or thetransferrin receptor are representative examples.

In another embodiment the Golgi retention signal of the alpha 1,3GalNAcT is disrupted by site-directed mutagenesis. This approach mutatesthe amino acids responsible for localizing the galactosyltransferase tothe Golgi compartment. The resultant glycosyltransferase is transportedto the plasma membrane where it becomes anchored via its modifiedtransmembrane sequences.

In vitro folding of alpha 1,3 GalNAcT requires proper disulfide bondformation. Ways to ensure proper disulfide bond formation includeS-sulfonation of the protein prior to disulfide formation, use ofoxido-shuffling reagents, and mutation of free Cys residue to Thr. Inthe in vitro folding of alpha 3GalNAc-T, the stem region acts as achaperone. Additionally, there are additives that can be used to preventthe hydrophobic collapse, including polyethylene glycol (PEG, e.g.PEG-4000) or L-arginine-HCl. PEG-4000 and L-arginine are thought tobeneficially affect the solubility of folding intermediates of bothcatalytic domain-proteins (CD-proteins) and stem region/catalytic domainproteins (SRCD-proteins) during in vitro folding or protein obtainedfrom inclusion bodies. In the case of catalytic domain (CD)-proteins,the majority of misfolded proteins are insoluble in the absence ofPEG-4000 and L-arginine and so they precipitate out during dialysis.Thus, the process will leave behind the properly folded molecules insolution bound to UDP-agarose that are enzymatically active.

When the catalytic domain of alpha 3GalNAc-T is expressed in E. Coli, itforms insoluble inclusion bodies. These inclusion bodies can becollected and then solubilized and folded in vitro to producecatalytically active domains. General methods for isolating and foldinginclusion bodies containing galactosyltransferase catalytic domains havebeen previously described (Ramakrishnan et al., J. Biol. Chem.,276:37665 (2001)). Thus, the in vitro folding efficiency is directlyrelated to the quantity of active enzyme that is produced from theisolated inclusion bodies. Accordingly, methods to increase the in vitrofolding efficiency would provide increased production of catalyticdomains that can be used to create useful products. US Application20060084162, incorporated by reference in its entirety herein, providesmaterials and methods that improve in vitro folding of catalytic domainsfrom galactosyltransferases that are related to the use of a stem regionof alpha 3GalNAc-T. Such methods are of use in the instant invention.

Methods of the Invention

The methods as described herein provide the ability to conjugatemultiple agents to compounds or compositions of the invention. Anembodiment of the present invention provides a glycoconjugate in whichone or more bioactive agents are bound to a modified saccharide residue,e.g., a modified galactose, which is in turn bound to a targetingcompound, e.g., a compound capable of binding a receptor on a cellmembrane. The 2′ modified galactose can be used as a handle to delivertherapeutic agents to specific tissue sites. In this manner, manytargeting glycoconjugates can be constructed. For example, a genedelivery system for genetic therapy can be produced by binding anucleotide and a ligand or antibody to the modified sugar. A therapeuticcompound for cancer can be produced by binding a chemotherapeutic agentand a ligand or antibody, e.g., an antibody to a cancer antigen, to themodified sugar residue.

The glycoconjugates can be manufactured as designer glycoconjugates,according to therapeutic need. As such, the designer polypeptide itselfcan be used for the targeting and drug delivery. The glycoconjugates canbe manufactured as nanoparticles. In certain examples, a biologicalsubstrate, such as a bioactive agent, for example a therapeutic agent,is used to engineer the nanoparticle. In other examples a second, third,fourth or more bioactive polypeptide is used in association with thenanoparticle to engineer multivalent nanoparticles. The bioactive agentsdo not have to be the same, for example a nanoparticle comprising threebioactive agents may comprise a chemotherapeutic, a tracking agent and atargeted delivery agent, such as an antibody.

Nanoparticles of the invention have use in methods of treating diseases.In other examples, the methods of the invention are used to engineer aglycoprotein from a magnetic resonance agent for use in diagnostictherapies. In these preferred examples, nanoparticles are engineered asdescribed herein, where the nanoparticles are superparamagneticnanoparticle.

Polypeptide fragments of the invention having altered donor and acceptorspecificity can be used to catalyze the linkage of numerous sugars froma donor to numerous acceptor sugars. Linkage of sugar derivatives canalso achieved through use of the altered catalytic domains of theinvention due to their expanded donor and acceptor specificity.

The presence of modified sugar moieties on a glycoprotein makes itpossible to link bioactive molecules via modified glycan chains, therebyassisting in the assembly of bionanoparticles that are useful fordeveloping the targeted drug delivery system and contrast agents forexample for use in imaging, e.g. magnetic resonance imaging. Thereengineered recombinant glycosyltransferases as described herein alsomake it possible to remodel the oligosaccharide chains of glycoproteindrugs, and to synthesize oligosaccharides for vaccine development.

Targeted Glycoconjugates

The alpha 1-3 N-Acetylgalactosaminyltransferases (alpha 3 GalNAc-T) asdescribed herein transfer a sugar from a sugar donor to a sugaracceptor. A sugar acceptor can be selected from galactose beta 1,4glcNac or galactose beta 1,4 glucose. Sugars that can be transferredinclude UDP-galactose, UDP-galactose analogues, UDP-GalNAc andUDP-GalNAc analogues. This reaction allows galactose to be linked to asugar acceptor, for example galactose beta 1,4 glcNAc or galactose beta1,4 glucose, that may itself be linked to a variety of other molecules,such as sugars and proteins, e.g., therapeutic agents, imaging agents,antibodies.

As described herein, modifications in sugar donors are tolerated by thealpha 3GalNAc enzymes. The alpha 3GalNAc enzymes of the invention havethe ability to use unnatural substrates (altered donor specificity) insugar transfer reactions due to altered donor specificities. The alpha 3GalNAc-T enzymes have a wider range of donor specificity, e.g. are ableto tolerate a wider range of donors, due to substitutions in thesugar-nucleotide binding pocket. For example, in certain embodiments asdescribed herein, substitutions in bovine a3Gal-T that broadens a3Gal-Tdonor specificity and makes the enzyme a3 GalNAc-T.

In certain examples, the one or more substitutions in the substratebinding site comprise an amino acid substitution at position 280, 281,or 282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or moresubstitutions in the substrate hinge region comprise an amino acidsubstitution at position 191 corresponding to bovine alpha 1,3galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In oneembodiment, the one or more substitutions close to the DXD motifcomprise an amino acid substitution at position 228 corresponding tobovine alpha 1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21).In another embodiment, wherein a serine (S) is substituted for ahistidine (H) at amino acid position 280, a glycine (G) is substitutedfor an alanine (A) at amino acid position 281, or a glycine (G) issubstituted for an alanine at amino acid position 282 of (SEQ ID NO 21).In another embodiment, a serine (S) or an alanine (A) is substituted fora proline (P) at amino acid position 191 corresponding to (SEQ ID NO:21). In still another embodiment, a glutamine (Q) is replaced with a amethionine (M) at amino acid position 228 of (SEQ ID NO:21).

In one embodiment of the invention, the donor sugar is modified so as toinclude a functional group at the C2 position of the sugar ring,preferably a ketone or an azido or a thiol functionality. In anotherembodiment, the modified sugar is a galactose which is modified at theC2 position by the addition of ketone functionality.

WO 2005/051429, incorporated by reference in its entirety herein,describes methods used to bind a bioactive agent to the modified sugar.The bioactive compounds may preferably include a functional group whichmay be useful, for example, in forming covalent bonds with the sugarresidue, which are not generally critical for the activity of thebioactive agent. Examples of such functional groups include, forexample, amino (—NH:2), hydroxy (—OH), carboxyl (—COOH), thiol (—SH),phosphate, phosphinate, ketone group, sulfate and sulfinate groups. Ifthe bioactive compounds do not contain a useful group, one can be addedto the bioactive compound by, for example, chemical synthetic means.Where necessary and/or desired, certain moieties on the components maybe protected using blocking groups, as is known in the art, see, e.g.,Green & Wuts, Protective Groups in Organic Synthesis (John Wiley &Sons)(1991).

Exemplary covalent bonds by which the bioactive compounds may beassociated with the sugar residue include, for example, amide (—CONH—);thioamide (—CSNH—); ether (ROR′, where R and R′ may be the same ordifferent and are other than hydrogen); ester (—COO—); thioester(—COS—); -0-; —S—; —Sn—, where n is greater than 1, preferably about 2to about 8; carbamates; —NH—; —NR—, where R is alkyl, for example, alkylof from about 1 to about 4 carbons; urethane; and substituted imidate;and combinations of two or more of these.

Covalent bonds between a bioactive agent and a modified sugar residuemay be achieved through the use of molecules that may act, for example,as spacers to increase the conformational and topographical flexibilityof the compound. Examples of such spacers include, for example, succinicacid, 1,6-hexanedioic acid, 1,8-octanedioic acid, and the like, as wellas modified amino acids, such as, for example, 6-aminohexanoic acid,4-aminobutanoic acid, and the like.

One of skill in the art can easily chose suitable compatible reactivegroups for the bioactive agent and the modified sugar, so as to generatea covalent bond between the bioactive agent and the modified sugar.Also, while the glycoconjugates of the invention are generally describedwith the targeting agent as the acceptor molecule or structure ontowhich a donor molecule (e.g., UDP-galactose) is actively linked throughthe action of a catalytic domain of a galactosyltransferase thebioactive agent can also be an acceptor molecule.

In certain embodiments, the instant method can be used to monitorglycosylation, for example the glycosylation of therapeuticglycoproteins and monoclonal antibodies. The potential ofglycosyltransferase enzymes to produce glycoconjugates carrying sugarmoieties with reactive groups may be a benefit to the glycotargeting ofdrugs to their site of action. Although a great number of pharmaceuticalagents are discovered each year, the clinical application of these ismany times hindered because of failure to reach the site of action. Themethods described herein that include using reengineeredglycosyltransferases to transfer chemically reactive sugar residues forlinking of other molecules via specific glycan chains may be used as anefficient drug delivery system.

Detection

The a3 GalNAc-T as described herein have application in the detection ofspecific sugar residues on a glycan chain of a glycoconjugates and inthe glycoconjugation and assembly of bio-nanoparticles for the targeteddelivery of bioactive agents. Protein glycoslation is one of the mostabundant posttranslational modifications and plays a fundamental role inthe control of biological systems and in disease.

Accordingly, glycosylation has been found to be a marker in disease.Additionally, carbohydrate modifications have been shown to be importantfor host-pathogen interactions, inflammation, development, andmalignancy (Varki, 1993; Lasky, 1996).

The methods described herein offer the advantages the modificationoccurs in a site directed manner, only where the carbohydrate isattached to the glycoprotein. Such specificity permits, for example, theuse of site-directed immunotherapy without affecting the antigen bindingaffinity of the immunoglobulin. Such specificity permits, further, thepotential use of this approach in developing a drug delivery system orbiological probes.

Imaging

Included in the invention are methods for imaging a target cell ortissue in a subject. The methods as described herein compriseadministering to a subject a polypeptide fragment synthesized by themethod comprising incubating a reaction mixture comprising a polypeptidefragment from a a3 GalNAc-T with a sugar donor, wherein one or moreimaging agents are linked to the sugar donor, and an sugar acceptorthereby imaging a target cell or tissue.

Coupling

Methods of transfer of C2 modified galactose analogues, for example C2keto galactose from its UDP derivative to the GlcNAc residue on theN-glycan chain of ovalbumin or to an asialo-agalacto-IgG1 molecule havebeen described in the art, for example in WO 2005/051429, incorporatedby reference in its entirety herein. The C2 modified galactoseanalogues, for example C2 keto galactose can be biotinylated, thusallowing for biotinylation of carriers such as ovalbumin and IgG.

The method of coupling a target agent to a carrier protein via glycanchains, for example ovalbumin and IgG1, is advantageous over othercross-linking methods. In the instant method, the target agent is linkedin a site-directed manner, only where the carbohydrate is attached tothe glycoprotein, for example as in the IgG1 molecule at the Fc domain,away from the antigen binding site. A problem encountered in previousapproaches using monoclonal antibodies for immunotherapy is the lack ofspecificity of the reactions, resulting in heterologous labeling and adecrease in the antibody affinity for the antigen. The instant inventionovercomes this problem.

Accordingly, the invention features methods of coupling an agent oragents to a carrier protein. The methods described herein comprisecoupling an agent to a carrier protein comprising incubating a reactionmixture comprising a polypeptide fragment of an alpha 1,3N-Acetylgalactosylaminotransferase (alpha 3GalNaC T) that retains theability to transfer a sugar with a chemically reactive functional groupfrom a sugar donor to a sugar acceptor, wherein the sugar donor iscoupled to an agent and the sugar acceptor is a carrier protein.

Thr polypeptide fragment can comprise in certain examples SEQ ID NO: 1,SEQ ID NO; 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO; 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, with a sugardonor, and a carrier protein, in the presence of manganese.

The sugar donor is, in certain examples, a UDP-galactose analogue, thatcan comprise an azido group, a keto group, or a thiol group. The azidogroup, the keto group or the thiol group can be substituted at the C2position of galactose, thus allowing for linking of agents. Accordingly,in certain preferred examples, one or more agents are linked to a sugarmoiety of the sugar donor. The agent can be selected from the groupconsisting of: antibodies, single chain antibodies, bacterial toxins,growth factors, therapeutic agents, targeting agents, contrast agents,chemical labels, a radiolabels, and fluorescent labels.

The carrier protein, in preferred examples, is ovalbumin. The carrierprotein, in other preferred examples, is an IgG. In certain instances,it is advantageous to couple the C2 UDP-galactose analogue to biotin fordetection. Subsequent detection of biotin can be carried out bychemiluminescent assay. The method as described herein is useful forimaging procedures, for example in magnetic resonance imaging.

Carbohydrate Synthesis

Enzymatic carbohydrate synthesis using glycosyltransferases is regio-and stereospecific and does not require extensive protecting groupdesigns. Naturally occurring carbohydrates have been successfullyprepared by this biomimetic pathway. The novelalpha(1-3)galactosylaminotransferases described herein have use in, forexample, the design and synthesis of natural and non-naturalcarbohydrate libraries for pharmaceutical purposes, for example, thesynthesis of sialyl-Lewis(a)- and sialyl-Lewis(x)-libraries. Baisch etal. (Carbohydr Res. 1998 November; 312(1-2): 61-72, incorporated byreference in its entirety herein).

Mimetics of a terminal tetrasaccharide region of many cellularglycoproteins and glycolipids (sialyl Lewis X) have demonstrated toinhibit angiogenesis both in vitro and in vivo and this may be used forcancer treatment

Antibodies and Applications

As described herein, the targeting compound may be an antibody or afragment thereof. The term“antibody” (Ab) or“monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody portions (e.g.,Fab and F (ab′)₂ portions and Fv fragments) which are capable ofspecifically binding to a cell surface marker. Such portions aretypically produced by proteolytic cleavage, using enzymes such as papain(to produce Fab portions) or pepsin (to produce F (ab′)₂ portions).Alternatively, antigen-binding portions can be produced through theapplication of recombinant DNA technology.

The immunoglobulin can be a “chimeric antibody” as that term isrecognized in the art. Also, the immunoglobulin may be a bifunction or ahybrid antibody, that is, an antibody which may have one arm having aspecificity for one antigenic site, such as a tumor associated antigen,while the other arm recognizes a different target, for example, a haptenwhich is, or to which is bound, an agent lethal to the antigen-bearingtumor cell. Alternatively, the bifunctional antibody may be one in whicheach arm has specificity for a different epitope of a tumor associatedantigen of the cell to be therapeutically or biologically modified. Inany case, the hybrid antibodies have a dual specificity, preferably withone or more binding sites specific for the hapten of choice or one ormore binding sites specific for a target antigen, for example, anantigen associated with a tumor, an infectious organism, or otherdisease state.

Biological bifunctional antibodies are described, for example, inEuropean Patent Publication, EPA 0 105 360, which is incorporated hereinby reference. Hybrid or bifunctional antibodies may be derivedbiologically, by cell fusion techniques, or chemically, especially withcross-linking agents or disulfide bridge-forming reagents, and may becomprised of those antibodies and/or fragments thereof. Methods forobtaining such hybrid antibodies are disclosed, for example, in PCTapplication WO83/03679, published Oct. 27, 1983, and published EuropeanApplication EPA 0 217 577, published Apr. 8, 1987, which areincorporated herein by reference. In one embodiment, the bifunctionalantibodies are biologically prepared from a polydome or a quadroma, orare synthetically prepared with cross-linking agents such asbis-(maleimideo)-methyl ether(“BMME”), or with other cross-linkingagents familiar to those skilled in the art.

In addition, the immunoglobin may be a single chain antibody (“SCA”).These may consist of single chain Fv fragments (“scFv”) in which thevariable light (“V [L]”) and variable heavy (“V [H]”) domains are linkedby a peptide bridge or by disulfide bonds. Also, the immunoglobulin mayconsist of single V [H] domains (dAbs) which possess antigen-bindingactivity. See, e.g., G. Winter and C. Milstein, Nature, 349: 295 (1991);R. Glockshuber et al., Biochemistry, 29: 1362 (1990); and, E. S. Ward etal., Nature, 341: 544 (1989).

The antibodies may, in certain embodiments, be chimeric monoclonalantibodies. As used herein, the term “chimeric antibody” refers to amonoclonal antibody comprising a variable region, i.e., binding region,from one source or species and at least a portion of a constant regionderived from a different source or species, usually prepared byrecombinant DNA techniques.

Chimeric antibodies comprising a murine variable region and a humanconstant region are preferred in certain applications of the invention,particularly human therapy, because such antibodies are readily preparedand may be less immunogenic than purely murine monoclonal antibodies.Such murine/human chimeric antibodies are the product of expressedimmunoglobulin genes comprising DNA segments encoding murineimmunoglobulin variable regions and DNA segments encoding humanimmunoglobulin constant regions. Other forms of chimeric antibodiesencompassed by the invention are those in which the class or subclasshas been modified or changed from that of the original antibody. Such“chimeric” antibodies are also referred to as “class-switchedantibodies.” Methods for producing chimeric antibodies involveconventional recombinant DNA and genetransfection techniques well knownin the art. See, e.g., Morrison, S. L. et al., Proc. Nat'l Acad. Sci.,81: 6851 (1984).

Encompassed by the term “chimeric antibody” is the concept of “humanizedantibody,” that is those antibodies in which the framework or“complementarity” determining regions (“CDR”) have been modified tocomprise the CDR of an immunoglobulin of different specificity ascompared to that of the parent immunoglobulin. (See, e.g., EPA 0 239 400(published Sep. 30, 1987)) In a preferred embodiment, a murine CDR isgrafted into the framework region of a human antibody to prepare the“humanized antibody.” See, e.g., L. Riechmann et al., Nature, 332: 323(1988); M. S, Neuberger et al., Nature, 314: 268 (1985). Furthermore,the immunoglobulin (antibody), or fragment thereof, used in the presentinvention may be polyclonal or monoclonal in nature. Monoclonalantibodies are the preferred immunoglobulins. The preparation of suchpolyclonal or monoclonal antibodies is well known to those skilled inthe art. See, e.g., G. Kohler and C. Milstein, Nature, 256: 495 (1975).The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the cell surfacemarker or an antigenic portion thereof can be administered to an animalin order to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of protein is preparedand purified so as to render it substantially free of naturalcontaminants. Such a preparation is then introduced into an animal inorder to produce polyclonal antisera of greater specific activity.However, the present invention should not be construed as limited inscope by any particular method of production of an antibody whetherbifunctional, chimeric, bifunctional-chimeric, humanized, or anantigen-recognizing fragment or derivative thereof.

In a preferred embodiment, the antibodies of the present invention aremonoclonal antibodies (or portions thereof). Such monoclonal antibodiescan be prepared using hybridoma technology (Kohler et al., Nature, 256:495 (1975); Kohler et al., Eur. J. Immunol., 6: 511 (1976); Kohler etal, Eur. J. Immunol., 6: 292 (1976); Hammerling et al., In: “MonoclonalAntibodies and T-Cell Hybridomas,” Elsevier, N.Y., pp. 563-681 (1981)).In general, such procedures involve immunizing an animal (preferably amouse) with a protein antigen or with a protein-expressing cell(suitable cells can be recognized by their capacity to bind antibody).The splenocytes of such immunized mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention. After fusion, theresulting hybridoma cells are selectively maintained in HAT medium, andthen cloned by limiting dilution as described by Wands et al.,Gastroenterology, 80: 225-232 (1981). The hybridoma cells obtainedthrough such a selection are then assayed to identify clones whichsecrete antibodies capable of binding the antigen. In addition,hybridomas and/or monoclonal antibodies which are produced by such

hybridomas and which are useful in the practice of the present inventionare publicly available from sources such as the American Type CultureCollection or commercial retailers.

The antibodies of the present invention may be labeled, for example, fordetection or diagnostic purposes, e.g., imaging. Labels for theantibodies of the present invention include, but are not limited to, thefollowing: examples of enzyme labels include malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcoholdehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholineesterase; examples of radioisotopic labels include 3H, 111In, 125I,131I, 32p, 35S, 14c, 51Cr, 57To, 58Co, 59Fe, 75Se, 152Eu, 90Y, 67Cu,217Ci, 211At, 212Pb, 47Sc, and 109Pd; examples of suitablenon-radioactive isotopic labels include 157Gd, 55Mn, 52Tr, and 56Fe;examples of fluorescent labels include an 152 Eu label, a fluoresceinlabel, an isothiocyanate label, a rhodamine label, a phycoerythrinlabel, aphycocyanin label, an allophycocyanin label, an o-phthaldehydelabel, and a fluorescamine label; examples of toxin labels includediphtheria toxin, ricin, and cholera toxin; examples of chemiluminescentlabels include a luminal label, an isoluminal label, an aromaticacridinium ester label, an imidazole label, an acridinium salt label, anoxalate ester label, a luciferin label, a luciferase label, and anaequorin label; and examples of nuclear magnetic resonance contrastingagents include heavy metal nuclei such as Gd, Mn, and Fe. Typicaltechniques for binding the above-described labels to antibodies areprovided by Kennedy et al., Clin. Chim Acta, 70:1-31 (1976), and Schurset al., Clin. Chim. Acta, 81: 1-40 (1977), which are incorporated byreference

In one embodiment, the glycoconjugates of the invention includemonoclonal antibodies, such as those directed against tumor antigens,for use as cancer therapeutics. Generally, monoclonal antibodies haveone N-linked bi-antennary oligosaccharide attached at the IgG-Fc region.The terminal sugars of the oligosaccharide moiety come in severalglycoforms, for example, some are desialated, degalactosylated, withonly terminal N-acetylglucosaminyl residues.

The monoclonal antibodies carrying only terminal N-acetylgucosamine onthe bi-antennary oligosaccharide moieties, the Goglycoform, can begenerated by de-sialylation and de-galactosylation of the monoclonalantibodies. With the Tyr289Leu-Gal-Tl (Y289LGalT1) enzyme andUDP-a-galactose-C-2-modified, a galactose moiety that has a chemicallyreactive group attached at the C2 position of galactose, can then betransferred to Go glycoform of the monoclonal antibody. The chemicallyreactive group can include, for example, a ketone moiety that can serveas a neutral, yet versatile chemical handle to add other agents, such asbioactive agents, to the compound.

Methods of Treatment

The instant invention provides enzymes and methods that can be used topromote the chemical linkage of biologically important molecules thathave previously been difficult to link, and thus provides a means tolink agents for therapeutic application. Moreover, the instant inventionprovides a means to carry out the method in a physiological setting.

Accordingly, the invention features methods for the diagnosis ortreatment of a subject suffering from a disease or disorder. The methodscomprise administering to the subject an effective amount of polypeptidefragment synthesized by the method comprising incubating a reactionmixture comprising an isolated catalytic domain from an alpha 1,3N-Acetylgalactosaminyltransferase (alpha3GalNac-T) with a sugar donor,wherein one or more agents are linked to the sugar donor, and an sugaracceptor thereby diagnosing or treating the subject.

In certain preferred embodiments, the polypeptide fragment is encoded bya nucleotide sequence that encodes an alpha 1,3N-Acetylgalactosaminyltransferase (alpha3GalNac-T) that transfers asugar with a chemically reactive functional group from a sugar donor toa sugar acceptor. In other certain preferred embodiments, thepolypeptide fragment is encoded by a nucleotide sequence which is atleast 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous tothe nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO; 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, or SEQ ID NO: 19 or a complement thereof.

Disease states needing treatment are only limited by current availabletherapeutics. As described herein, the methods of the invention areuseful for engineering of nanoparticles, including multivalentnanoparticles, carrying any number of therapeutic agents. For example,the nanoparticles can be used to treat cancer, inflammatory disease,cardiovascular disease, obesity, ageing, bacterial infection, or anyother disease amenable to therapy.

The glycoconjugates compositions of the invention can be used to treatand/or diagnose a variety of diseases and/or disorders. For example, theglycoconjugates compositions of the invention are used for specific,targeted delivery of bioactive agents, including toxic drugs, agents forimaging or diagnostics, (e.g., toxins, radionuclides), totherapeutically-relevant tissues or cells of the body, for example,tumors. In another embodiment of the invention, the glycoconjugatescompositions of the invention are used to deliver bioactive agents,including DNA vectors, to cells.

As further examples, the glycoconjugates compositions of the inventionare useful for the treatment of a number of diseases and/or disordersincluding, but not limited to: cancer, both solid tumors as well asblood-borne cancers, such as leukemia; hyperproliferative disorders thatcan be treated by the compounds of the invention include, but are notlimited to, neoplasms located in the: abdomen, bone, breast, digestivesystem, liver, pancreas, peritoneum, endocrine glands (adrenal,parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, headand neck, nervous (central and peripheral), lymphatic system, pelvic,skin, soft tissue, spleen, thoracic, and urogenital.

The glycoconjugates of the invention can be used to treat cardiovasculardiseases and disorders including, but not limited to, myocardialinfarction (heart attack), cerebrovascular diseases (stroke), transientischaemic attacks (TIA), peripheral vascular diseases, arteriosclerosis,angina, high blood pressure, high cholesterol, arrhythmia.

The glycoconjugates of the invention can be used to treat geneticdiseases, such as enzyme deficiency diseases.

The glycoconjugates of the invention can be used to treathyperproliferative disorders. Examples of such hyperproliferativedisorders that can be treated by the glycoconjugates of the inventionare as described in Application WO 2005/051429, and are incorporated byreference in its entirety herein.

The glycoconjugates of the present invention are also useful for raisingan immune response against infectious agents. Viruses are one example ofan infectious agent that can cause disease or symptoms that can betreated by the compounds of the invention. Examples of viruses that cancause disease or symptoms and that can be treated by the glycoconjugatesof the invention are as described in Application WO 2005/051429, and areincorporated by reference in its entirety herein.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated by the glycoconjugates of the invention are asdescribed in Application WO 2005/051429, and are incorporated byreference in its entirety herein.

Additionally, the glycoconjugates of the invention are useful fortreating autoimmune diseases. An autoimmune disease is characterized bythe attack by the immune system on the tissues of the victim. Autoimmunedisease is characterized by the inability of the recocognition of “self”and the tissue of the afflicted subject is treated as a foreign target.The compounds of the present invention are therefore useful for treatingautoimmune diseases by desensitizing the immune system to these selfantigens by provided a TCR signal to T cells without a costimulatorysignal or with an inhibitory signal. Examples of autoimmune diseaseswhich may be treated using the glycoconjugates of the present inventionare as described in Application WO 2005/051429, and are incorporated byreference in its entirety herein.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by glycoconjugates of the invention. Moreover, theglycoconjugates of the invention can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

The glycoconjugates of the invention which can inhibit an immuneresponse are also useful for treating and/or preventing organ rejectionor graft versus host disease, atherosclerosis; olitis; regionalenteritis; adult respiratory distress syndrome; local manifestations ofdrug reactions, such as dermatitis, etc.; inflammation-associated orallergic reaction patterns of the skin; atopic dermatitis and infantileeczema; contact dermatitis; psoriasis; lichen planus; allergicenteropathies; allergic rhinitis; bronchial asthma; hypersensitivity ordestructive responses to infectious agents; poststreptococcal diseases,e.g. cardiac manifestations of rheumatic fever, and the like.

Vaccines

The invention also provides methods for eliciting an immune response ina mammal such as a human, including administering to a subject animmunological composition comprising a compound or composition asdescribed herein. Therefore, one embodiment of the present invention isto use the glycoconjugates described herein in an immunologicalpreparation.

The immunological composition according to the instant invention may beprepared by any method known in the art. For example, glycoconjugates ofthe present invention are prepared and are then injected into anappropriate animal. The compositions according to the present inventionmay be administered in a single dose or they may be administered inmultiple doses, spaced over a suitable time scale to fully utilize thesecondary immunization response. For example, antibody titers may bemaintained by administering boosters once a month. The vaccine mayfurther comprise a pharmaceutically acceptable adjuvant, including, butnot limited to Freund's complete adjuvant, Freund's incomplete adjuvant,lipopolysaccharide, monophosphoryl lipid A, muramyl dipeptide, liposomescontaining lipid A, alum,muramyl tripeptide-phosphatidylethanoloamine,keyhole and limpet hemocyanin.

Administration

The compositions of the present invention may be administered by anymeans that results in the contact of the bioactive agent with theagent's site or site (s) of action on or in a subject, e.g., a patient.The compositions may be administered alone or in conjunction with one ormore other therapies or treatments.

The targeted glycoconjugates produced according to the presentinvention, can be administered to a mammalian host by any route. Thus,as appropriate, administration can be orally, intravenously, rectally,parenterally, intracistemally, intradermally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, creams,drops or transdermal patch), bucally, or as an oral or nasal spray. Theterm “parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrastemal,subcutaneous and intraarticular injection and infusion. Parenteraladministration in this respect includes administration by the followingroutes: intravenous, intramuscular, subcutaneous, intraocular,intrasynovial, transepithelial including transdermal, ophthalmic,sublingual and buccal; topically including ophthalmic, dermal, ocular,rectal and nasal inhalation via insufflation, aerosol and rectalsystemic.

In addition, administration can be by periodic injections of a bolus ofthe therapeutic or can be made more continuous by intravenous orintraperitoneal administration from an external source. In certainembodiments, the therapeutics of the instant invention can bepharmaceutical-grade and incompliance with the standards of purity andquality control required for administration to humans. Veterinaryapplications are also within the intended meaning as used herein.

The formulations, both for veterinary and for human medical use, of thetherapeutics according to the present invention typically include suchtherapeutics in association with a pharmaceutically acceptable carriertherefor and optionally other ingredient (s). The carrier (s) can beacceptable in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.Pharmaceutically acceptable carriers are intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch asethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide.

Useful solutions for oral or parenteral administration can be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences. Formulations forparenteral administration also can include glycocholate for buccaladministration, methoxysalicylate for rectal administration, or citricacid for vaginal administration. The parenteral preparation can beenclosed in ampoules, disposable syringes or multiple dose vials made ofglass or plastic.

Formulations of the present invention suitable for oral administrationcan be in the form of discrete units such as capsules, gelatin capsules,sachets, tablets, troches, or lozenges, each containing a predeterminedamount of the drug; in the form of a powder or granules; in the form ofa solution or a suspension in an aqueous liquid or non-aqueous liquid;or in the form of an oil-in-water emulsion or a water-in-oil emulsion.The therapeutic can also be administered in the form of a bolus,electuary or paste. A tablet can be made by compressing or molding thedrug optionally with one or more accessory ingredients. Compressedtablets can be prepared by compressing, in a suitable machine, the drugin a free-flowing form such as a powder or granules, optionally mixed bya binder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding, in a suitable machine, a mixtureof the powdered drug and suitable carrier moistened with an inert liquiddiluent.

Oral compositions generally include an inert diluent or an ediblecarrier.

For the purpose of oral therapeutic administration, the active compoundcan be incorporated with excipients. Oral compositions prepared using afluid carrier for use as a mouthwash include the compound in the fluidcarrier and are applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gumtragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition can be sterile and can be fluid to the extentthat easy syringability exists. It can be stable under the conditions ofmanufacture and storage and can be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, and sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization, e.g., filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, methods ofpreparation include vacuum drying and freeze-drying which yields apowder of the active ingredient plus any additional desired ingredient.

Formulations suitable for topical administration, including eyetreatment, include liquid or semi-liquid preparations such as liniments,lotions, gels, applicants, oil-in-water or water-in-oil emulsions suchas creams, ointments or pasts; or solutions or suspensions such asdrops. Formulations for topical administration to the skin surface canbe prepared by dispersing the therapeutic with a dermatologicallyacceptable carrier such as a lotion, cream, ointment or soap. In someembodiments, useful are carriers capable of forming a film or layer overthe skin to localize application and inhibit removal.

For inhalation treatments, such as for asthma, inhalation of powder(self-propelling or spray formulations) dispensed with a spray can, anebulizer, or an atomizer can be used. Such formulations can be in theform of a finely comminuted powder for pulmonary administration from apowder inhalation device or self-propelling powder-dispensingformulations. In the case of self-propelling solution and sprayformulations, the effect can be achieved either by choice of a valvehaving the desired spray characteristics (i.e., being capable ofproducing a spray having the desired particle size) or by incorporatingthe active ingredient as a suspended powder in controlled particle size.For administration by inhalation, the therapeutics also can be deliveredin the form of an aerosol spray from a pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer. Nasal drops also can be used.

Systemic administration also can be by transmucosal ortransdermal means.For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants generally are known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and filsidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. Fortransdermal administration, thetherapeutics typically are formulated into ointments, salves, gels, orcreams as generally known in the art.

The therapeutics can be prepared with carriers that will protect againstrapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.

The compounds of the invention may also suitably be administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58, 481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U. Sidman etal., Biopolymers 22: 547-556 (1983)), poly (2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and R.Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R.Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release compositions also include liposomally entrappedcompositions of the present invention (Epstein, et al., Proc. Natl.Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA 77: 4030-4034 (1980).

The compositions can be formulated in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Generally, the therapeutics identified according to the invention can beformulated for administration to humans or other mammals, for example,in therapeutically effective amounts, e.g., amounts which provideappropriate concentrations of the bioactive agent to target tissue/cellsfor a time sufficient to induce the desired effect. Additionally, thetherapeutics of the present invention can be administered alone or incombination with other molecules known to have a beneficial effect onthe particular disease or indication of interest. By way of exampleonly, useful cofactors include symptom-alleviating cofactors, includingantiseptics, antibiotics, antiviral and antifungal agents and analgesicsand anesthetics.

The effective concentration of the therapeutics identified according tothe invention that is to be delivered in a therapeutic composition willvary depending upon a number of factors, including the final desireddosage of the drug to be administered and the route of administration.The preferred dosage to be administered also is likely to depend on suchvariables as the type and degree of the response to be achieved; thespecific composition of another agent, if any, employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thecomposition; the duration of the treatment; bioactive agent (such as achemotherapeutic agent) used in combination or coincidental with thespecific composition; and like factors well known in the medical arts.In some embodiments, the therapeutics of this invention can be providedto an individual using typical dose units deduced from theearlier-described mammalian studies using non-human primates androdents. As described above, a dosage unit refers to a unitary, i.e. asingle dose which is capable of being administered to a patient, andwhich can be readily handled and packed, remaining as a physically andbiologically stable unit dose comprising either the therapeutic as suchor a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

Therapeutics of the invention also include “prodrug” derivatives. Theterm prodrug refers to a pharmacologically inactive (or partiallyinactive) derivative of a parent molecule that requiresbiotransformation, either spontaneous or enzymatic, within the organismto release or activate the active component. Prodrugs are variations orderivatives of the therapeutics of the invention which have groupscleavable under metabolic conditions. Prodrugs become the therapeuticsof the invention which are pharmaceutically active in vivo, when theyundergo solvolysis under physiological conditions or undergo enzymaticdegradation. Prodrug forms often offer advantages of solubility, tissuecompatibility, or delayed release in the mammalian organism (see,Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp.352-401, Academic Press, San Diego, Calif., 1992).

Therapeutic or Diagnostic Agents

A wide variety of agents may be included in the compounds of the presentinvention, such as any biologically active, therapeutic or diagnosticcompound or composition. In general, the term bioactive agent includes,but is not limited to: polypeptides, including proteins and peptides(e.g., insulin); releasing factors and releasing factor inhibitors,including Luteinizing Hormone Releasing Hormone (LHRH) and gonadotropinreleasing hormone (GnRH) inhibitors; carbohydrates (e.g., heparin);nucleic acids; vaccines; and pharmacologically active agents such asanti-infectives such as antibiotics and antiviral agents; anti-fungalagents; analgesics and analgesic combinations; anesthetics; anorexics;anti-helminthics; anti-arthritic agents; respiratory drugs, includinganti-asthmatic agents and drugs for preventing reactive airway disease;anticonvulsants; antidepressants; anti-diabetic agents; anti-diarrheals;anticonvulsants; antihistamines; anti-inflammatory agents; toxins,anti-migraine preparations; anti-nauseants; anticancer agents, includinganti-neoplastic drugs; anti-parkinsonism drugs; anti-pruritics;anti-psychotics; antipyretics; antispasmodics; anticholinergics;sympathomimetics; xanthine derivatives; cardiovascular preparationsincluding potassium and calcium channel blockers, beta-blockers,alpha-blockers, cardioprotective agents; anti-arrhythmics;anti-hyperlipidemic agents; anti-hypertensives; diuretics;anti-diuretics; receptor agonists, antagonists, and/or mixed functionagonist/antagonists; vasodilators including general coronary, peripheraland cerebral; central nervous system stimulants; vasoconstrictors; coughand cold preparations, including decongestants; enzyme inhibitors;hormones such as estradiol, testosterone, progesterone and othersteroids and derivatives and analogs, including corticosteroids;hypnotics; hormonolytics; immunosuppressive agents; muscle relaxants;parasympatholytics; central nervous system stimulants; diuretics;hypnotics leukotriene inhibitors; mitotic inhibitors; muscle relaxants;genetic material, including nucleic acid, RNA, DNA, recombinant RNA,recombinant DNA, antisense RNA, antisense DNA, hammerhead RNA, aribozyme, a hammerheadribozyme, an antigene nucleic acid, aribo-oligonucleotide, a deoxyribonucleotide, an antisenseribo-oligonucleotide, and/or an antisense deoxyribo-oligonucleotide;psychostimulants; sedatives; anabolic agents; vitamins; herbal remedies;anti-metabolic agents; anxiolytics; attention deficit disorder (ADD) andattention deficit hyperactivity disorder (ADHD) drugs; neuroleptics; andtranquilizers.

Application No. WO 2005/051429, incorporated by reference in itsentirety herein, provides a list of exemplary agents that can beconjugated to the compositions of the instant invention.

Kits

Also included in the invention are kits. Preferably, kits comprise apackaging material, and a polypeptide fragment from an alpha 1,3N-Acetylgalactosaminyltransferase (alpha3GalNac-T) according to any oneof the aspects of the invention as described herein. The kits, incertain preferred embodiments, comprise a sugar donor. The donor can beany one of UDP-galactose, UDP-GalNAc, UDP-GalNAc analogues orUDP-Galactose analogues. The kits can also comprise an agent. Inpreferred examples, the agent is linked to the sugar donor. Exemplaryagents are described in this disclosure. Certain agents can be selectedfrom antibodies, single chain antibodies, bacterial toxins, growthfactors, therapeutic agents, contrast agents, targeting agents, chemicallabels, a radiolabels, and fluorescent labels.

EXAMPLES

It should be appreciated that the invention should not be construed tobe limited to the examples that are now described; rather, the inventionshould be construed to include any and all applications provided hereinand all equivalent variations within the skill of the ordinary artisan.

As described in more detail below, the experiments reported herein arebased on the structure-based design of a 1,3N-Acetylgalactosaminyltransferase (a3 GalNac-T) from a 1,3galactosyltransferase (a3 Gal-T) which was mutated at seven positions tobroaden a3Gal-T donor specificity and make it a3GalNac-T.

Example 1 Mutation of a3Gal-T Broadens a3Gal-T Donor Specificity andMakes it a3GalNac-T

X-ray crystal structures of the catalytic domain of manyglycosyltransferases have been determined in recent years, and thesestudies show that the specificity of the sugar donor is determined byresidues in the sugar-nucleotide binding pocket of glycosyltransferases.This structural information has made it possible to reengineer theexisting glycosyltransferases.

Described herein is the stricture based design of an alpha1-3-N-acetylgalactosaminyltransferase from a1-3-galactosyltransferase.FIG. 1 is a schematic showing the structure—based design ofa1-3-N-acetylgalactosaminyltransferase from a1-3-galactosyltransferase.In FIG. 2, the sugar sugar donor binding site and the hinge region wherethe substitutions occur are shown. These regions are the regions wherethe substitutions occur in the a1-3-galactosyltransferase. Mutation ofresidues in these regions leads to the novel alpha 1-3GalNAc-transferases described herein that can transfer 2′-modifiedgalactose.

FIG. 3 and FIG. 4 shows transfer of UDP-modified sugars by the alpha 1,3Gal-T-191A . . . 280SGG282 enzyme. FIG. 5 is a Table showing the effectof substitutions in the donor substrate binding site, hinge region andnear DXD motif on Gal activity, GalNAc activity and GalKeto activity isshown.

Methods

The invention was performed using the following methods:

Met344His Mutant

Site-directed mutagenesis was performed using the PCR method.Construction of the enzymes was carried out as described previously inQasba et al. (Biochemistry 2004, 43, 12513-12522), incorporated byreference in its entirety herein.

Bacterial Growth and Plasmid Transformation

Bacterial growth and plasmid transformations can be performed usingstandard procedures (Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley-Interscience, New York(1987)). US Published Application 20060084162, incorporated by referencein its entirety herein, describes methods for bacterial growth andtransformation using the plasmid pEGT-d129, which encodes the catalyticdomain (residues 130-402) of bovine .beta.(1,4)-galactosyltransferase I.Site-directed mutagenesis can be performed using a CLONTECHsite-directed mutagenesis transformer kit. Thus, the transformationmixture contains the template pEGT-d129, a selection primer, and amutagenic primer for creation of a desired enzyme. Enzymes are screenedfor the incorporated substitutions by looking for changes in restrictionenzyme digestion patterns and confirmed by DNA sequencing. The positiveclones were transformed into B834(DE3)pLysS cells.

Expression and Purification of Inclusion Bodies

The expression and purification of the inclusion bodies can be carriedout as described previously (Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-Interscience,New York (1987)). The inclusion bodies are S-sulfonated by dissolving in5 M GdnHCl, 0.3 M sodium sulfite, and the addition of di-sodium2-nitro-5-thiosulfobenzoate to a final concentration of 5 mM. Thesulfonated protein is precipitated by dilution with water, and theprecipitate was washed thoroughly.

Briefly, 100 mg of sulfonated protein is folded in one liter foldingsolution for 48 hours. Inclusion of 10% glycerol and 10 mM lactose inthe folding solution enhances the folding efficiency of thegalactosyltransferase, e.g. beta-1,4-galactosyltransferase(beta4Gal-T1). After refolding the protein, the folding solution isextensively dialyzed against water. During dialysis the misfoldedprotein precipitates out, while the folded protein remains soluble. Thesoluble protein is first concentrated and then purified on a Ni-columnNearly 2 mg of folded ppGalNAc-T2 protein is obtained form 1 liter offolding solution. Purified protein may be tested for catalytic activityusing a 13 amino acid peptide, PTTDSTTPAPTTK, as an acceptor usingmethods described previously (Fritz. T. A et al. J Biol. Chem. 2006).

Improving the folding conditions: In recent years factorial foldingscreens (Rudolph and Lilie, FASEB J., 10:40-56 (1996); Chen and Gouaux,Proc. Natl. Acad. Sci., 94:13431-13436 (1997); Armstrong et al., Prot.Sci., 8:1475-1483 (1999)) have been developed for examining the foldingefficiencies of proteins from inclusion bodies. To improve the in vitrofolding efficiency, 8 different folding conditions similar to theformulations described in the Foldlt Screen kit (Hampton Research,Calif.) with certain modifications were tested. Condition I: 50 mMTris-HCl pH 8.0, 5 mM EDTA, 0.5 M guanidine-HCl, 8 mM cysteamine and 4mM cystamine. Condition II: 55 Mes pH 6.5, 10.56 mM NaCl, 0.44 mM KCl,2.2 mM MgCl.sub.2, 2.2 mM CaCl.sub.2, 0.5 M guanidine-HCl. ConditionIII: similar to condition II with respect to the buffer, pH, chaotropeand salt condition, but it had 0.055% PEG-4000, 1.1 mM EDTA, 0.44 Msucrose and 0.55 M L-arginine. Condition IV: 55 mM Mes pH 6.5, 264 mMNaCl, 11 mM KCl, 0.055% PEG-4000, 0.5 M guanidine-HCl, 2.2 mM MgCl₂, 2.2mM CaCl.sub.2 and 0.44 M sucrose. Condition V: 55 mM Tris pH 8.2, 10.56mM NaCl, 0.44 mM KCl, 1.1 mM EDTA, 0.44 M sucrose. Conditions VI andVIII are similar except for the presence of redox agents. Condition VII:55 mM Mes pH 6.5, 264 mM NaCl, 11 mM KCl, 1.1 mM EDTA, 0.5 Mguanidine-HCl, and 0.55 M L-arginine. The buffers II through VII had 100mM GSH and 10 mM GSSG. Conditions I and VIII, had 8 mM cysteamine and 4mM cystamine. Condition VIII, gave the highest enzymatic activity,soluble and folded protein, was 50 mM Tris-HCl pH 8.0, 10.56 mM NaCl,0.44 mM KCl, 2.2 mM MgCl₂, 2.2 mM CaCl.sub.2 0.5 M guanidine-HCl, 8 mMcysteamine and 4 mM cystamine, 0.055% PEG-4000 and 0.55 M L-arginine.

Mutation

Mutation of certain amino acid residues as described herein is, incertain examples, performed site-directed mutagenesis. US PublishedApplication 20060084162 describes methods for site directed mutagensisof amino acid position 289 of the bovine.beta.(1,4)-galactosyltransferase I, performed using the PCR method. Themethod is easily adapted by one of skill in the art to the instantinvention for engineering of 1,3 N-Acetylgalactosylaminotransferase(alpha 3GalNaC T) enzymes from the alpha Gal T.

Gal-T and GalNAc-T Enzyme Assays

Gal-T and GalNAc T enzyme assays are easily performed according tomethods described in the art, for example US Published Application20060084162. Protein concentrations are measured using the Bio-Radprotein assay kit, based on the method of Bradford and further verifiedon SDS gel. An in vitro assay procedure for the Gal-T1 has been reportedpreviously (Ramakrishnan et al., J. Biol. Chem., 270, 87665-376717(2001)). The activities were measured using UDP-Gal or UDP-GalNAc assugar nucleotide donors, and GlcNAc and Glc as the acceptor sugars. Forthe specific activity measurements, a 100-.mu.l incubation mixturecontaining 50 mM .beta.-benzyl-GlcNAc, 10 mM MnCl.sub.2, 10 mM Tris-HCl,pH 8.0, 500 .mu.M UDP-Gal or UDP-GalNAc, 20 ng of Gal-T1, and 0.5 .mu.Clof [.sup.3H]UDP-Gal or [.sup.3H]UDP-GalNAc was used for each Gal-T orGalNAc-T reaction. The incubation was carried out at 37.degree. C. for10 mM The reaction was terminated by adding 200 .mu.l of cold 50 mMEDTA, and the mixture was passed through a 0.5-ml bed volume column ofAG1-X8 cation resin (Bio-Rad) to remove any unreacted [.sup.3H]UDP-Galor [.sup.3H]UDP-GalNAc. The column was washed successfully with 300,400, and 500 .mu.l of water, and the column flow-through was dilutedwith Biosafe scintillation fluid; radioactivity was measured with aBeckman counter. A reaction without the acceptor sugar was used as acontrol. A similar assay was carried out to measure the GalNAc-Tactivity with Glc and other acceptors in the presence of 50 .mu.M bovineLA (Sigma).

The in vitro assay for enzyme activity (beta Gal T1, double mutantbeta-gal) can be performed as described (Boeggeman et al., Glycobiology,12:395-407 (2002)). The .sup.3H-labeled-UDP-Gal or UDP-Galactose wasused as sugar donor and GlcNAc as the sugar acceptor. A reaction withoutGlcNAc was used as a control.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

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What is claimed is:
 1. A method of coupling an agent to a carrierprotein comprising: incubating a reaction mixture comprising a bioactiveagent, a carrier protein and a polypeptide fragment of a mutated alpha1,3 N-acetylgalactosaminyltransferase (alpha 3GalNAc T) that is amutated form of a 1,3 galactosyltransferase (alpha 3 Gal-T) comprisingone or more mutations in the sugar donor binding site or hinge region ofthe alpha 3 Gal-T and that retains the ability to transfer a donor sugarwith a chemically reactive functional group to a sugar acceptor, whereinsaid sugar acceptor is attached to the carrier protein and wherein saiddonor sugar is transferred to said sugar acceptor and said transferreddonor sugar is then coupled to the bioactive agent having a group thatreacts with said chemically reactive functional group, thereby formingan oligosaccharide that couples said bioactive agent to said carrierprotein.
 2. The method of claim 1, wherein said oligosaccharide is atrisaccharide.
 3. The method of claim 2, wherein the trisaccharide isselected from the group consisting of GalNAc alpha1-3-Gal beta 1-4GlcNAcand 2′-modified-Gal alpha 1-3-Gal beta 1-4GlcNAc.
 4. The method of claim1, wherein said polypeptide fragment comprises any one of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO; 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:
 20. 5. Themethod of claim 1, wherein said chemically reactive functional group ofsaid donor sugar is selected from the group consisting of a ketone, anazido and a thiol functionality.
 6. The method of claim 1, wherein saiddonor sugar is a galactose which is modified at the C2 position by theaddition of ketone functionality.
 7. The method of claim 1, wherein saiddonor sugar is a galactose which is modified by the addition of an azidogroup.
 8. The method of claim 1, wherein said agent is selected from thegroup consisting of a single chain antibody, a bacterial toxin, a growthfactor, a therapeutic agent, a targeting agent, a contrast agent, achemical label, a radiolabel, and a fluorescent label.
 9. The method ofclaim 1, wherein said carrier protein is selected from the groupconsisting of ovalbumin and IgG1.